Fluidic connectors and microfluidic systems

ABSTRACT

Fluidic connectors, methods, and devices for performing analyses (e.g., immunoassays) in microfluidic systems are provided. In some embodiments, a fluidic connector having a fluid path is used to connect two independent channels formed in a substrate so as to allow fluid communication between the two independent channels. One or both of the independent channels may be pre-filled with reagents (e.g., antibody solutions, washing buffers and amplification reagents), which can be used to perform the analysis. These reagents may be stored in the channels of the substrate for long periods amounts of time (e.g., 1 year) prior to use.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/467,653, filed May 9, 2012, issued as U.S. Pat. No. 8,409,527, andentitled “Fluidic Connectors and Microfluidic Systems”, which is acontinuation of U.S. patent application Ser. No. 12/113,503, filed May1, 2008 and issued as U.S. Pat. No. 8,202,492, and entitled “FluidicConnectors and Microfluidic Systems”, which claims priority under 35U.S.C. §119(e) to U.S. Provisional Patent Application No. 60/927,640,filed May 4, 2007, and entitled “Fluidic Connectors and MicrofluidicSystems”, each of which is incorporated herein by reference.

FIELD OF INVENTION

The present invention relates generally to microfluidic systems andcomponents thereof, and more specifically, to fluidic connectors,methods, and devices for performing analyses in microfluidic systems.

BACKGROUND

The delivery of fluids plays an important role in fields such aschemistry, microbiology and biochemistry. These fluids may includeliquids or gases and may provide reagents, solvents, reactants, orrinses to chemical or biological processes. While various microfluidicdevices and methods, such as microfluidic assays, can provideinexpensive, sensitive and accurate analytical platforms, fluid deliveryto the platform can add a level of cost and sophistication, as theoperation of microfluidic devices often requires the ability to exchangefluids between the device itself and the outside world. In some cases,the operation of the device includes one or a combination of thefollowing: introduction of a sample, introduction of reagents,extraction of a fluid for off-chip analysis or transfer of fluids fromone chip to the next.

Because microfluidic devices benefit from scaling law, most applicationsrequire only minute quantities of fluid to carry out assays, compared totheir bench-top counterparts. Along with the development of theseminiaturized systems, the microfluidic community has invested manyefforts in designing interfaces between the microfluidic device and thelaboratory world. The major problem associated with world-to-chipconnection is the mismatch between the volumes used on-chip (e.g.,femtoliters to microliters) with respect to the volumes typicallyhandled at the bench (e.g., microliters to liters). For instance, manyworld-to-chip connectors have dead volume, e.g., wasted volume that maylie at the core of the connector itself. For example, in the case of atubing with a small inner diameter (e.g., 200 μm to inject smallquantities of fluid) connecting to a microchannel (e.g., 10-200 μm indiameter), there may remain a gap between the edge of the tubing and theentrance of the microchannel. The volume defined by that gap is referredto as dead volume, and, in some instances, can be of the same order ofmagnitude as the total volume of sample to be analyzed. In practice, thedead volume of many devices can often be higher than the volume ofsample analyzed by the chip; this is an undesired effect forapplications that rely on small sample/reagent consumption.

Accordingly, advances in the field that could, for example, reduce thedead volume and/or allow easy interface between the microfluidic systemand the user would be beneficial.

SUMMARY OF THE INVENTION

Fluidic connectors, methods, and devices for performing analyses (e.g.,immunoassays) in microfluidic systems are provided.

In one aspect of the invention, a series of devices are provided. In oneembodiment, a device includes a microfluidic system formed in asubstrate comprising a first microfluidic channel including at least oneinlet and one outlet and a second microfluidic channel including atleast one inlet and one outlet. The device also includes a fluidicconnector that can be connected to the substrate. The fluid connectorcomprises a fluid path including a fluid path inlet and a fluid pathoutlet, wherein upon connection, the fluid path inlet connects to theoutlet of the first microfluidic channel to allow fluid communicationbetween the fluid path and the first microfluidic channel, and the fluidpath outlet connects to the inlet of the second microfluidic channel toallow fluid communication between the fluid path and the secondmicrofluidic channel. The fluid path may contain a reagent (e.g., one ormore fluids such as a sample or a series of reagents) disposed thereinprior to connection of the fluidic connector to the substrate. In somecases, the microfluidic system is constructed and arranged to operatewithout recirculation of a fluid in the system.

In some embodiments, the first and second microfluidic channels are notin fluid communication with one another prior to first use, and at firstuse, the first and second microfluidic channels are brought into fluidcommunication with one another.

The first microfluidic channel may comprise a first reagent disposedtherein prior to connection of the fluidic connector to the substrate.The first microfluidic channel may further comprise a second reagentdisposed therein prior to connection of the fluidic connector to thesubstrate. The first and second reagents may be fluid reagents separatedby a fluid immiscible with said first and second reagents. The first andsecond reagents may be, for example, liquid reagents and the fluidimmiscible with said first and second reagents may be a gas. In someembodiments, the second microfluidic channel contains a reagent disposedtherein. The reagent in the second microfluidic channel may be driedprior to first use, and, in some cases, is adsorbed to a surface of thesecond microfluidic channel. The device may further comprises a cover(e.g., a tape) positioned adjacent the substrate so as to enclose thefirst and second microfluidic channels.

In some embodiments, the device further comprising a reaction area influid communication with the first and/or second microfluidic channels,wherein the reaction area allows detection of a chemical and/orbiological reaction in the reaction area. The reaction area may compriseat least one meandering channel region. In some cases, the reaction areacomprises at least two meandering channel regions connected in series.The at least two meandering channel regions can comprises a chemicaland/or biological species that can undergo a chemical and/or biologicalreaction. Each of the at least two meandering channel regions may allowformation and/or detection of a single, homogenous signal in each ofsaid regions upon carrying out a chemical and/or biological reaction insaid regions.

The device may further comprising a first detector aligned with thefirst meandering channel region. In some embodiments, the devicecomprises a second detector aligned with the second meandering channelregion.

The microfluidic system may include any suitable numbers of channelintersections; for example, the system may include less than 2 channelintersections. In one embodiment, the microfluidic system does notinclude any channel intersections.

In certain embodiments, the fluid path has a first volume and furthercomprises a volume control element that can allow introduction of acontrolled volume of fluid less than the first volume into the fluidpath prior to connection of the fluidic connector to the microfluidicsystem. The fluidic connector may comprise at least one non-fluidicfeature complementary to a feature of the substrate so as to form anon-fluidic connection between the fluidic connector and the substrateupon connection. The fluidic connector may comprise at least one featurecomplementary to a feature of the substrate so as to form anirreversible connection between the fluidic connector and the substrate.The fluidic connector can further comprise a sampling element that canreceive a fluid sample from a biological entity. The fluidic connectormay allow transfer of fluid from the biological entity to the fluidpath.

In some cases, the device further comprises a source of vacuum that canbe connected to an outlet.

In another embodiment, a device comprises a microfluidic system formedin a substrate comprising a first microfluidic channel including aninlet and an outlet and a second microfluidic channel including an inletand an outlet. The device also includes a fluidic connector that can beconnected to the substrate comprising a fluid path including a fluidpath inlet and a fluid path outlet, wherein upon connection, the fluidpath inlet connects to the outlet of the first microfluidic channel andthe fluid path outlet connects to the inlet of the second microfluidicchannel. The fluidic connector further comprises at least onenon-fluidic feature complementary to a feature of the substrate so as toform a non-fluidic connection between the fluidic connector and thesubstrate upon connection.

In some cases, the microfluidic system is constructed and arranged tooperate without recirculation of a fluid in the system. In someembodiments, the first and second microfluidic channels are not in fluidcommunication with one another prior to first use, and at first use, thefirst and second microfluidic channels are brought into fluidcommunication with one another.

In another embodiment, a device comprises a first microfluidic channelformed in a substrate and containing a first reagent disposed therein,and a second microfluidic channel formed in the substrate and containinga second reagent disposed therein, wherein the first and secondmicrofluidic channels are not in fluid communication with one anotherprior to first use, and at first use, the first and second microfluidicchannels are brought into fluid communication with one another. Thefirst microfluidic channel may further comprise a third reagent, thefirst and third reagents being separated by a fluid immiscible with saidreagents. The second reagent is dried prior to first use.

In another embodiment, a device comprises a microfluidic system formedin a substrate comprising a first microfluidic channel including aninlet and an outlet and a second microfluidic channel including an inletand an outlet. The device also includes a fluidic connector that can beconnected to the substrate and may comprise a fluid path including afluid path inlet and a fluid path outlet, wherein upon connection, thefluid path inlet connects to the outlet of the first microfluidicchannel to allow fluid communication between the fluid path and thefirst microfluidic channel, and the fluid path outlet connects to theinlet of the second microfluidic channel to allow fluid communicationbetween the fluid path and the second microfluidic channel. The fluidpath further comprises a volume control element that can allowintroduction of a controlled volume of fluid less than the first volumeinto the fluid path prior to connection of the fluidic connector to themicrofluidic system. In some cases, the volume control element is afrit.

In another embodiment, a device comprises a microfluidic system formedin a substrate comprising a first microfluidic channel including aninlet and an outlet and a second microfluidic channel including an inletand an outlet. The device also includes a fluidic connector that can beconnected to the substrate and may comprise a fluid path including afluid path inlet and a fluid path outlet, wherein upon connection, thefluid path inlet connects to the outlet of the first microfluidicchannel and the fluid path outlet connects to the inlet of the secondmicrofluidic channel. The fluidic connector further comprises a samplingelement that can puncture a biological component. The biologicalcomponent may be human skin. The sampling element may be used to receivea fluid sample from the biological component. The fluidic connector mayallow transfer of fluid from the biological entity to the fluid path.

In another aspect of the invention, a series of methods is provided. Inone embodiment, a method of storing reagents comprises positioning afirst reagent in a first microfluidic channel formed in a substrate andpositioning a second reagent in a second microfluidic channel formed inthe substrate, wherein the first and second microfluidic channels arenot in fluid communication with one another during the positioningsteps. The method also includes sealing an inlet and/or outlet of thefirst microfluidic channel so as to store the first reagent in the firstmicrofluidic channel, and sealing an the inlet and/or outlet of thesecond microfluidic channel so as to store the second reagent in thesecond microfluidic channel.

In some embodiments, prior to sealing, the first microfluidic channelcontains a third reagent disposed therein, the first and third reagentsseparated by a fluid immiscible with said reagents. The second reagentmay be dried prior to sealing of the inlet of the second microfluidicchannel.

In another embodiment, a method comprises providing a first microfluidicchannel formed in a substrate and containing a first reagent disposedtherein prior to first use, and providing a second microfluidic channelformed in the substrate and containing a second reagent disposed thereinprior to first use. The first and second microfluidic channels are notin fluid communication with one another prior to first use, and whereinat first use, the first and second microfluidic channels are broughtinto fluid communication with one another. The method also includescausing the first and second microfluidic channels to be in fluidcommunication with one another.

In some embodiments, the causing step comprises connecting a fluid pathbetween the first and second microfluidic channels. The fluid path maycontains a sample disposed therein. The sample may be, for example, afluid sample.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIGS. 1A and 1B are schematic diagrams of a microfluidic deviceincluding a fluidic connector according to an embodiment of theinvention;

FIG. 2 is a block diagram of a microfluidic system that may containstored reagents and can be used for performing an a chemical and/orbiological reaction according to an embodiment of the invention;

FIGS. 3A-3D are schematic diagrams of a microfluidic device including afluidic connector and containing stored reagents used to perform achemical and/or biological reaction according to an embodiment of theinvention;

FIGS. 4A-4D are schematic diagrams of a microfluidic device including afluidic connector and containing stored reagents used to perform achemical and/or biological reaction according to an embodiment of theinvention;

FIGS. 5A-5F are photographs of a microfluidic device including a fluidicconnector used to perform a chemical and/or biological reactionaccording to an embodiment of the invention;

FIG. 6 is a block diagram of a microfluidic system according to anembodiment of the invention;

FIGS. 7A-7D are schematic diagrams of a microfluidic device that can beused with an open-ended fluidic device to perform a chemical and/orbiological reaction according to an embodiment of the invention;

FIGS. 8A-8D are schematic diagrams of an open-ended fluidic device andfluidic connectors according to an embodiment of the invention;

FIGS. 9A-9F are schematic diagrams of monolithic fluidic connectorsaccording to an embodiment of the invention;

FIGS. 10A and 10B are schematic diagrams of another fluidic connectoraccording to an embodiment of the invention;

FIGS. 11A and 11B are schematic diagrams of fluidic connectors that canbe connected orthogonally or on the same plane as the channels of amicrofluidic system according to an embodiment of the invention;

FIGS. 12A-12E are schematic diagrams of a fluidic connector includingclips that can be used to attach the fluidic connector to a substrateaccording to an embodiment of the invention;

FIG. 13 is a schematic diagram of features that can be included on, forexample, a fluidic connector and/or a substrate to secure attachmentbetween the connector and substrate according to an embodiment of theinvention;

FIGS. 14A and 14B are schematic diagrams showing a perspective view of adevice including an alignment element, a fluidic connector, and asubstrate according to an embodiment of the invention;

FIGS. 15A and 15B are schematic diagrams showing a cross-sectional viewof a device including an alignment element that and a substrate that areformed of a single part according to an embodiment of the invention;

FIGS. 16A and 16B are schematic diagrams showing a cross-sectional viewof a device including an alignment element that and a substrate that areformed of separate parts according to an embodiment of the invention;

FIGS. 17A-17C are schematic diagrams of a device including detectionzones in the form of meandering regions according to an embodiment ofthe invention;

FIGS. 18A and 18B are schematic diagrams of an optical system fordetecting a component in a detection zone of a device according to anembodiment of the invention;

FIG. 19 is a schematic diagram of an optical system for detectingcomponents in different detection zones of a device according to anembodiment of the invention;

FIG. 20 is a schematic diagram of an optical system including an opticallight source and a detector aligned with each detection zone of a deviceaccording to an embodiment of the invention.

DETAILED DESCRIPTION

Fluidic connectors, methods, and devices for performing analyses (e.g.,immunoassays) in microfluidic systems are provided. In some embodiments,a fluidic connector having a fluid path is used to connect twoindependent channels formed in a substrate so as to allow fluidcommunication between the two independent channels. One or both of theindependent channels may be pre-filled with reagents (e.g., antibodysolutions, washing buffers and amplification reagents), which can beused to perform the analysis. These reagents may be stored in thechannels of the substrate for long periods amounts of time (e.g., 1year) prior to use. Prior to connection of the fluid connector and thesubstrate, the fluid path may be filled with a sample (e.g., blood). Thesample may be obtained, for example, by pricking a finger of a useruntil blood is drawn from the finger into the fluid path (e.g., bycapillary forces). Upon connection of the fluidic connector and thechannels of the substrate, the sample can pass through a reaction areawithin the first channel of the substrate. This process can allowcomponents of the sample to interact with components disposed in thereaction area. Afterwards, reagents from the second channel can flow tothe reaction area via the fluid path, allowing components in thereaction area to be processed (e.g., amplified to produce detectablesignal). Components in the reaction area can then be determined usingvarious methods of detection.

Microfluidic systems described herein may be useful for performingchemical and/or biological reactions, especially immunoassays, with oneor more advantages such as: (a) use of small amounts of sample withlittle or no sample waste, (b) long-term stability of chemical and/orbiological reagents stored in the device, (c) reduction ofcross-contamination between stored reagents and/or between sample andreagent, (d) sample metering, (e) ease of use to untrained users forintroducing a sample into the device, (f) efficient mixing of reagents,and (g) assay reliability. These and other advantages are described inmore detail below in connection with the description and figures.

The articles, systems, and methods described herein may be combined withthose described in International Patent Publication No. WO2005/066613(International Patent Application Serial No. PCT/US2004/043585), filedDec. 20, 2004 and entitled “Assay Device and Method,” InternationalPatent Publication No. WO2005/072858 (International Patent ApplicationSerial No. PCT/US2005/003514), filed Jan. 26, 2005 and entitled “FluidDelivery System and Method,” and International Patent Publication No.WO2006/113727 (International Patent Application Serial No.PCT/US06/14583), filed Apr. 19, 2006 and entitled “Fluidic StructuresIncluding Meandering and Wide Channels,” each of which is incorporatedherein by reference in its entirety.

FIG. 1 shows a microfluidic device 10 according to one embodiment of theinvention. As shown in this illustrative embodiment, device 10 comprisestwo attachable units: substrate 20, which includes a microfluidic system22, and a fluidic connector 40, which can be used to connect twoindependent microfluidic channels of the substrate. Microfluidic system22 of substrate 20 includes channel 24 having an inlet 26 and an outlet28, as well as channel 34 having an inlet 36 and an outlet 38. As shownin the illustrative embodiment of FIG. 1A, channels 24 and 34 are notconnected; that is, there is no fluid communication between thechannels. As described in more detail below, non-connected channels maybe advantageous in certain cases, such as for storing different reagentsin each of the channels. For example, channel 24 may be used to storedry reagents and channel 34 may be used to store wet reagents. Havingthe channels be physically separated from one another can enhancelong-term stability of the reagents stored in each of the channels,e.g., by keeping the reagent(s) stored in dry form protected frommoisture that may be produced by reagent(s) stored in wet form.

As shown, fluidic connector 40 includes a fluid path 42 having an inlet46 and an outlet 44. Fluidic connector 40 can be connected to substrate20, e.g., via the inlets and outlets. Upon connection, fluid path inlet46 connects to outlet 38 of microfluidic channel 34 and fluid pathoutlet 44 connects to inlet 26 of microfluidic channel 24. Thisconnection causes fluid communication between channels 24 and 34 viafluid path 42. The connections between the inlets and outlets of thearticle and the substrate may form fluid-tight seals to prevent leakageat the points of connection. Accordingly, as illustrated in FIG. 1B, iffluid flows in the direction of arrow 56, at least a portion of a fluidin channel 34 can flow into fluid path 42 and then into channel 24,optionally exiting at outlet 28.

Although FIG. 1A shows only two separate channels forming microfluidicsystem 22, in other embodiments, a microfluidic system may include morethan two separate channels, and a fluidic connector can be used toconnect three or more such channels of a substrate. In such embodiments,a fluidic connector may have multiple fluid paths (which may beinterconnected or independent) and/or multiple inlets and/or outletsthat can connect to several different microfluidic channels of thesubstrate. Additionally, although FIG. 1 shows two separate channels 24and 34 on the same substrate, article 40 can be used to connect channelson different substrates.

The microfluidic system formed by the connection of two independentchannels of a substrate using a fluidic connector, as shown in FIG. 1B,is an example of an “open-loop” system. As used herein, an “open-loop”system is constructed and arranged to operate without recirculation of afluid within the microfluidic system. In other words, a fluid portionstarting out at a first position within the microfluidic system does notpass the first position again after it leaves that position. Instead,the fluid portion may exit the device at an outlet (unless, for example,the fluid portion gets processed or used up in the microfluidic system).For example, as illustrated in FIG. 1B, a fluid portion initially atposition “A” and flowing in the direction of arrow 56 may flow intofluid path 42 and then into channel 24, optionally exiting at outlet 28;however, the design of the microfluidic system does not allow the fluidportion to re-enter channel 34 and to pass though position “A” again.Similarly, a fluid portion initially at position “B” and flowing in thedirection of arrow 56 may exit outlet 28; this fluid portion cannotenter into channel 34 or 24 to allow the portion to pass though position“B” again. In some cases, the microfluidic system does not allowrecirculation of a fluid within the system (e.g., during intended use).

In other embodiments, a fluidic connector can be used to form a“closed-loop” system. As used herein, a “closed-loop” system may allowrecirculation of a fluid within the microfluidic system such that afluid portion starting out at a first position within the microfluidicsystem can pass the first position again after it leaves that position.For example, if a second fluidic connector (e.g., one similar to fluidicconnector 40) was used to connect inlet 36 and outlet 28 of substrate 20of FIG. 1B, a closed-loop system would be formed. Alternatively, ifmicrofluidic system 22 was designed so that inlet 36 and outlet 28 werejoined such that channels 24 and 34 formed a single continuous channel,the connection of fluidic connector 40 to inlet 38 and outlet 26 wouldform a closed-loop system.

It should also be understood that a device described herein may includemore than one fluidic connector. Multiple fluid connectors are usefulfor connecting multiple channels (or portions of channels) of one ormore substrates.

In certain embodiments, a fluidic connector may be used to connect two(or more) portions of a single microfluidic channel of a substrate. Itshould be understood that where at least first and second separate(independent) channels of a substrate are described herein, a fluidicconnector may be used to connect similar embodiments but where at leasta portion of the first channel is in fluid communication with at least aportion of the second channel (e.g., to form a single interconnectedchannel) prior to connection using the fluidic connector.

Optionally, and as described in more detail below, fluidic connector 40may include at least one non-fluidic feature 52 complementary to afeature 54 of the substrate so as to form a non-fluidic connectionbetween the fluidic connector and the substrate upon connection of thefluid path. This non-fluidic connection can help to stabilize theconnection between the fluidic connector and substrate.

In some embodiments, fluidic connector 40 can be used to introduce oneor more fluids (e.g., a sample such as blood, serum, plasma, tear fluid,saliva, urine, sperm, sputum, or any other fluid of interest such as abuffer) into the microfluidic system of substrate 20. This can allow thesample (or other fluid) to bypass at least one channel of the substrate.For example, if a sample is first introduced into fluid path 42 and thenfluidic connector 40 is connected to substrate 20 as shown in FIG. 1B,flow of the fluids in the direction of arrow 56 allows the samplecontained in fluid path 42 to flow into channel 24, but not channel 34.Such a design may be useful for cases in which the sample to bedelivered via fluid path 42 contaminates or otherwise undesirablyaffects one or more components within channel 34. It should beunderstood, however, that the fluidic connector need not be used tointroduce fluids into the device, but can be used, in some embodiments,simply to fluidly connect at least two channels of a device or devices.

As described above, a fluid may be introduced into fluid path 42 viainlet 46 (or outlet 44, which may act as an inlet for purposes of fluidintroduction). All or a portion of fluid path 42 may be filled with thefluid. Optionally, fluidic connector 40 may include a secondary flowpath 48, which connects inlet 50 to flow path 42. This design can allow,for example, the introduction of a fluid into fluid flow path 42 viainlet 50 and secondary path 48 before or after the fluidic connector hasbeen connected to the substrate (e.g., as shown in FIG. 1B).Alternatively, a fluid can be introduced into fluid path 42 via inlet 50prior to connection of the fluidic connector and the substrate. In someembodiments, inlet 50 and secondary fluid path 48 can be blocked (e.g.,with a plunger or using any other suitable method) after introducingfluid into fluid path 42 via inlet 50 and secondary fluid path 48. Thisblocking can decrease the number of channel intersections of themicrofluidic system during operation of the device, and may beadvantageous for reasons described below.

In some embodiments, microfluidic systems described herein (includingdevice substrates and fluidic connectors) contain stored reagents priorto first use of the device and/or prior to introduction of a sample intothe device. The use of stored reagents can simplify use of themicrofluidic system by a user, since this minimizes the number of stepsthe user has to perform in order to operate the device. This simplicitycan allows microfluidic systems described herein to be used by untrainedusers, such as those in point-of-care settings. Stored reagents inmicrofluidic devices are particularly useful for devices designed toperform immunoassays.

FIG. 2 shows a block diagram 60 of a microfluidic device that maycontain stored reagents and can be used for performing an a chemicaland/or biological reaction (e.g., an immunoassay). The microfluidicdevice includes a reagent inlet 62 in fluid communication with a reagentstorage area 64, which may include, for example, one or more channelsand/or reservoirs. The device may also include a sample loading area 66,such as a fluidic connector that can connect reagent storage area 64 toreaction area 68. The reaction area, which may include one or more areasfor detecting a component in a sample (e.g., detection zones), may be influid communication with waste area 70 and coupled to outlet 72. In someembodiments, reaction area 68 is an immunoassay area.

In the exemplary embodiment shown in FIG. 2, section 80 comprises thereagent inlet and reagent storage area, and section 82 comprises thereaction area, waste area, and outlet. Reagents may be stored in one orboth of sections 80 and 82. For example, in one particular embodiment, areagent is stored in the form of a fluid (e.g., a liquid or a gas) inreagent storage area 64 of section 80, and a reagent in the form of adry film is stored in reaction area 68 of section 82.

In some embodiments, sections 80 and 82 are in fluid communication withone another (e.g., via sample loading area 66) prior to introduction ofa sample into the device. For example, if sample loading area 66included fluidic connector 40 of FIG. 1, the fluidic connector can beconnected to the substrate to cause fluid communication between sections80 and 82. Subsequently, a sample may be introduced into the device viainlet 50 and secondary fluid path 48.

In other embodiments, sections 80 and 82 are not in fluid communicationwith one another prior to introduction of a sample into the device. Forexample, if sample loading area 66 included fluidic connector 40 of FIG.1 which did not have inlet 50 or secondary fluid path 48, the fluidicconnector may first be filled with a sample and then connected to thesubstrate to cause fluid communication between sections 80 and 82. Inthis example, the sample is introduced into the channels of thesubstrate at the time when (or shortly after) fluid communication isformed between sections 80 and 82. In such instances, sections 80 and 82are not in fluid communication with one another prior to first use ofthe device, wherein at first use, the sections are brought into fluidcommunication with one another.

As described herein, one or more reagents that may be used in a chemicaland/or biological reaction may be stored and/or disposed in the device(e.g., in a device substrate and/or a fluid connector) prior to firstuse and/or prior to introduction of a sample into the device. Suchreagents may be stored and/or disposed in fluid and/or dry form, and themethod of storage/disposal may depend on the particular application.Reagents can be stored and/or disposed, for example, as a liquid, a gas,a gel, a plurality of particles, or a film. The reagents may bepositioned in any suitable portion of a device, including, but notlimited to, in a channel, reservoir, on a surface, and in or on amembrane, which may optionally be part of a reagent storage area. Areagent may be associated with a microfluidic system (or components of asystem) in any suitable manner. For example, reagents may be crosslinked(e.g., covalently or ionically), absorbed, or adsorbed (physisorbed)onto a surface within the microfluidic system. In one particularembodiment, all or a portion of a channel (such as a fluid path of afluid connector or a channel of the device substrate) is coated with ananti-coagulant (e.g., heparin). In some cases, a liquid is containedwithin a channel or reservoir of a device prior to first use and/orprior to introduction of a sample into the device.

In some embodiments, dry reagents are stored in one section of amicrofluidic device and wet reagents are stored in a second section of amicrofluidic device. Alternatively, two separate sections of a devicemay both contain dry reagents and/or wet reagents. The first and secondsections may be in fluid communication with one another prior to firstuse, and/or prior to introduction of a sample into the device, in someinstances. In other cases, the sections are not in fluid communicationwith one another prior to first use and/or prior to introduction of asample into the device. During first use, a stored reagent may pass fromone section to another section of the device. For instance, a reagentstored in the form of a fluid can pass from a first section to a secondsection of the device after the first and second sections are connectedvia a fluid path (e.g., a fluidic connector). In other cases, a reagentstored as a dried substance is hydrated with a fluid, and then passesfrom the first section to the second section upon connection of thesections. In yet other cases, a reagent stored as a dried substance ishydrated with fluid, but does not pass from one section to anothersection upon connection of the sections. Methods of storing reagents aredescribed in further detail below.

It should be understood that while the microfluidic system presented byblock diagram 60 includes only two sections 80 and 82, a microfluidicdevice may include additional sections in other embodiments.Additionally, the sequence of fluid flow between reagent storage area64, sample loading area 66, and reaction area 68 may be different insome devices. For example, fluid flow may be directed from a reagentstorage area to reaction area followed by fluid flow from a sampleloading area to the reaction area. Other arrangements are also possible.

FIGS. 3A-3D show an example of a microfluidic device including a fluidicconnector and containing stored reagents that can be used in a chemicaland/or biological reaction. Device 100 includes a first section 106including reagent storage area 110, which is in the form of a channel112 and includes an inlet 116 and an outlet 118. Different reagents maybe stored in channel 112 depending on the particular application. Forexample, if the device were used to perform an immunoassay, the channelmay have stored therein, in series, a rinse fluid 120, an antibody fluid122, a rinse fluid 124, a labeled-antibody fluid 126, and a rinse fluid128. Additional reagents and rinse fluids may also present as needed.These reagents may be in the form of plugs (e.g., liquid plugs) that areseparated from one another by immiscible fluid plugs 130 (e.g., aseparation fluid such as a gas (e.g., air, nitrogen, or argon) or an oil(e.g., a fluorocarbon or hydrocarbon)). In FIG. 3A, inlet 116 and outlet118 are sealed so as to prevent evaporation and contamination of thestored reagents.

Device 100 also includes a second section 150 having an inlet 154, anoutlet 156, a channel 158, reaction area 160, and a waste area 174. Thereaction area may include several detection zones 162, 164, 166, and168. The detection zones may have any suitable configuration and/orarrangement. In one embodiment, each of the detection zones is in theform of a meandering (serpentine) channel, as described in more detailbelow and in International Patent Publication No. WO2006/113727(International Patent Application Serial No. PCT/US06/14583), filed Apr.19, 2006 and entitled “Fluidic Structures Including Meandering and WideChannels,” which is incorporated herein by reference in its entirety.The detection zones may be arranged to detect, for example, differentcomponents of sample, or may be used as positive and/or negativecontrols. In some cases, one or more of the detection zones contains areagent stored therein. In one particular embodiment, a device used forperforming an immunoassay includes a series of stored dry reagents. Thereagents may be physisorbed onto a surface of the meandering channel.For example, detection zone 162 may include a negative control (e.g., adetergent known to prevent adhesion of proteins), detection zones 164and 166 may include different concentrations of antibodies that may bindto a component in a sample (or two different antibodies that can bind todifferent components in the sample), and detection zone 168 may includea positive control (e.g., the same antigen expected to be determinedfrom a sample). The positive control may be used as a qualitativecontrol; for example, if a signal reaches a certain threshold, the testcan be considered valid. Additionally and/or alternatively, the positivecontrol can also be as a quantitative tool; for example, the intensityof the signal can be can be part of an on-chip calibration process.

As shown in the embodiment illustrated in FIG. 3A, each of the areaswithin section 150 are in fluid communication with one another, but noneare in fluid communication with any of the components of section 106. Incertain embodiments, section 150 containing stored dry reagents andsection 106 containing stored wet reagents are configured to not be influid communication with one another prior to first use because thisconfiguration can promote long-term storage of each of the reagents intheir respective sections, as described further below.

As shown in FIG. 3B, sections 106 and 150 can be connected using fluidicconnector 178, causing sections 106 and 150 to be in fluid communicationwith one another. If outlet 118 and inlet 154 are covered with a seal(e.g., a biocompatible tape) in FIG. 3A, this connection can cause thesealings over the outlet and inlet to be pierced, broken, or removed.

Fluidic connector 178 may be used for sample loading and may includesample 180 contained therein. As described herein, sample 180 may beintroduced into fluidic connector 178 by an suitable method, and, insome cases, is introduced into the fluidic connector prior to therebeing fluid communication between sections 106 and 150.

As shown in the embodiment illustrated in FIG. 3C, fluids in reagentstorage area 110 and sample 180 may flow from section 106 towardssection 150. Fluid flow may take place, for example, by applying apositive pressure to inlet 116 (e.g., using a plunger, gravity, or apump) or by applying a vacuum source to outlet 156. In such embodiments,a source of positive pressure and/or vacuum may be connected to one ormore inlet(s) and/or outlet(s), respectively.

Sample 180 first flows into reaction area 160 (FIG. 3C), and then intowaste area 174 (FIG. 3D). The passing of the sample through thedetection zones allows interaction (e.g., binding) between one or morecomponents of the sample (e.g., an antigen) and one or more componentsin the reaction area (e.g., an antibody). As described herein, thecomponent(s) of the reaction area may be in the form of dried reagentsstored in the reaction area prior to first use. This interaction mayform a product such as a binding pair complex. In some cases, thisinteraction alone causes a signal to be determined (e.g., measured) by adetector coupled to the microfluidic system. In other cases, in orderfor an accurate signal to be determined by the detector, the product istreated by one or more reagents from reagent storage area 110. Forexample, a reagent stored in reagent storage area 110 may be alabeled-antibody that interacts with an antigen of the sample. Thisinteraction can allow the product to be labeled or the signal from theproduct to be amplified.

In one particular embodiment involving an immunoassay, the storedreagents in the storage area include an enzyme amplification solutionand a precipitating dye (e.g., diaminobenzidine, DAB). The one or morereagents from reagent storage area 110 is allowed to pass through eachof the detection zones. These reagents may interact further with abinding pair complex, e.g., to amplify the signal and/or to label thecomplex, as depicted in detection zones 164 and 168 of FIG. 3D.

By maintaining an immiscible fluid (a separation fluid) between each ofthe reagents in the reagent storage area, the stored fluids can bedelivered in sequence from the reagent storage area while avoidingcontact between any of the stored fluids. Any immiscible fluid thatseparates the stored reagents may be applied to the reaction areawithout altering the conditions of the reaction area. For instance, ifantibody-antigen binding has occurred at one of the detection zones ofthe reaction area, air can be applied to the site with minimal or noeffect on any binding that has occurred.

As described herein, storing reagents in a microfluidic system can allowthe reagents to be dispensed in a particular order for a downstreamprocess (e.g., amplifying a signal in a reaction area). In cases where aparticular time of exposure to a reagent is desired, the amount of eachfluid in the microfluidic system may be proportional to the amount oftime the reagent is exposed to a downstream reaction area. For example,if the desired exposure time for a first reagent is twice the desiredexposure time for a second reagent, the volume of the first reagent in achannel may be twice the volume of the second reagent in the channel. Ifa constant pressure differential is applied in flowing the reagents fromthe channel to the reaction area, and if the viscosity of the fluids isthe same or similar, the exposure time of each fluid at a specificpoint, such as a reaction area, may be proportional to the relativevolume of the fluid. Factors such as channel geometry, pressure orviscosity can also be altered to change flow rates of specific fluidsfrom the channel.

Additionally, this strategy of storing reagents in sequence, especiallyamplification reagents, can be adapted to a wide range of chemistries.For example, various amplification chemistries that produce opticalsignals (e.g., absorbance, fluorescence, glow or flashchemiluminescence, electrochemiluminescence), electrical signals (e.g.,resistance or conductivity of metal structures created by an electrolessprocess) or magnetic signals (e.g., magnetic beads) can be used to allowdetection of a signal by a detector.

The use of gaseous (e.g., air) plugs to separate reagents requires theoverall microfluidic device to be compatible with many air bubbles.Although air bubbles may be stabilized and/or controlled withinmicrofluidic devices using a variety of methods, one particular methodused in certain embodiments described herein includes limiting thenumber of channel intersections in the system. Accordingly, microfluidicdevices described herein may be designed to have few (e.g., less than 5,4, 3, or 2), one, or no channel intersections. As used herein, a channelintersection includes at least three channels (or portions of one ormore channels) intersecting at a single point (e.g., forming a “Y”). Forexample, device 100 of FIG. 3 does not have any channel intersectionsand device 200 of FIG. 4 has only one channel intersection 219. Devicesthat do not have any channel intersections may be useful, for example,for performing reactions that do not require mixing of reagents (e.g.,stored reagents).

FIGS. 4A-4D show another example of a microfluidic device including afluidic connector and containing stored reagents that can be used in achemical and/or biological reaction. As shown in these illustrativeembodiments, device 200 includes a first section 202 comprising reagentstorage area 204. The reagent storage area has two parts: upper portion205 and lower portion 206. The upper portion includes channel 208 havinginlet 216 connected thereto and channel 209 having inlet 217 connectedthereto. Channels 208 and 209 are separated in the upper portion andmeet at intersection 219, which is connected to channel 212 of the lowerportion. Channel 212 is connected to an outlet 218. Device 200 havingtwo inlets 216 and 217, each connected to a different channel, may beuseful, for example, for performing reactions in which two reagents needto be stored separately on the device, but which require mixing duringuse or immediately before use.

In one particular embodiment, device 200 is used to perform animmunoassay for human IgG, which uses sliver enhancement for signalamplification. A solution of silver salts is stored in channel 208 and asolution of hydroquinone is stored in channel 209. Because these twocomponents, which can produce signal amplification upon mixing, arelocated in separate channels, they cannot mix with each other until theflow drives both solutions towards intersection 219.

Reagents that do not have to be mixed with one another can be stored inlower portion 206 of the reagent storage area. These reagents caninclude, for example, rinse fluids, antibody fluids, and other fluids asneeded. The reagents may be in the form of plugs that are separated fromone another by immiscible fluid plugs 230 (e.g., a separation fluid suchas a gas (e.g., air) or an oil). In FIG. 4A, inlets 216 and 217, andoutlet 218 are sealed so as to prevent evaporation and contamination ofthe stored reagents.

Device 200 also includes a second section 250 having an inlet 254, anoutlet 256, a channel 258, a reaction area 260, and a waste area 274.The reaction area may include several detection zones 262, 264, 266, and268. Optionally, one or more detection zones may be in the form of ameandering channel region, as described herein. The detection zones maybe arranged to detect, for example, different components of sample, orused as positive and/or negative controls. In some cases, one or more ofthe detection zones contains a reagent stored therein. In oneembodiment, a device used for performing an immunoassay includes aseries of stored dry reagents. The reagents may be physisorbed onto asurface of a meandering channel of a detection zone.

In one particular embodiment, wherein device 200 is used for performingan immunoassay for human IgG and uses sliver enhancement for signalamplification, one or more surfaces of the meandering channels of thereaction area is modified by biomolecules such as BSA (bovine serumalbumin) or Tween, a negative control (e.g., a detergent known toprevent adhesion of proteins), different concentrations of antibodies(e.g., anti-human IgG) that may bind to a component in a sample, andhuman IgG, a positive control (e.g., the same antigen expected to bedetermined from a sample). These reagents are stored in section 250prior to use by sealing inlet 254 and outlet 256.

As shown in FIG. 4B, sections 202 and 250 can be connected using fluidicconnector 278, causing sections 202 and 250 to be in fluid communicationwith one another. Fluidic connector 278 may be used for sample loadingand may include sample 280 (e.g., blood) contained therein. As describedherein, sample 280 may be introduced into fluidic connector 278 by ansuitable method, and, in some cases, is introduced into the fluidicconnector prior to there being fluid communication between sections 202and 250.

As shown in the embodiment illustrated in FIG. 4C, fluids in reagentstorage area 204 and sample 280 may flow towards section 250. Fluid flowmay take place, for example, by applying a positive pressure to inlets216 and 217 (e.g., using a plunger, gravity, or a pump) or by applying avacuum source to outlet 256. Sample 280 first flows into reaction area260 (FIG. 4C), and then into waste area 274 (FIG. 4D). The passing ofthe sample through the detection zones allows interaction (e.g.,binding) between one or more components of the sample and one or morecomponents stored in the reaction area. This interactions may form, forexample, a product such as a binding pair complex. Subsequent flow offluids from the reagent storage area over the detection zones can causelabeling of the product and/or signal amplification.

In one particular embodiment, device 200 is used for performing animmunoassay for human IgG and uses sliver enhancement for signalamplification. After delivery of a sample containing human IgG from thefluidic connector to the reaction area, binding between the human IgGand a stored dry reagent, anti-human IgG, can take place. This bindingcan form a binding pair complex in a detection zone. Stored reagentsfrom lower portion 206 of reagent storage area 204 can then flow overthis binding pair complex. One of the stored reagents may include asolution of metal colloid (e.g., a gold conjugated antibody) thatspecifically binds to the antigen to be detected (e.g., human IgG). Thismetal colloid can provide a catalytic surface for the deposition of anopaque material, such as a layer of metal (e.g., silver), on a surfaceof the detection zone. The layer of metal can be formed by using a twocomponent system as described above: a metal precursor (e.g., a solutionof silver salts), which can be stored in channel 208, and a reducingagent (e.g., hydroquinone), which can be stored in channel 209. As apositive or negative pressure differential is applied to the system, thesilver salt and hydroquinone solutions eventually merge at intersection219, where they mix slowly (e.g., due to diffusion) along channel 212,and then flow over the reaction area. Therefore, if antibody-antigenbinding occurs in the reaction area, the flowing of the metal precursorsolution through the area can result in the formation of an opaquelayer, such as a silver layer, due to the presence of the catalyticmetal colloid associated with the antibody-antigen complex. The opaquelayer may include a substance that interferes with the transmittance oflight at one or more wavelengths. Any opaque layer that is formed in themicrofluidic channel can be detected optically, for example, bymeasuring a reduction in light transmittance through a portion of thereaction area (e.g., a meandering channel) compared to a portion of anarea that does not include the antibody or antigen. Alternatively, asignal can be obtained by measuring the variation of light transmittanceas a function of time, as the film is being formed in a detection zone.The opaque layer may provide an increase in assay sensitivity whencompared to techniques that do not form an opaque layer.

FIGS. 5A-5F show images of a device used to perform a human IgGimmunoassay according to one embodiment of the invention, and isdescribed in more detail in the Examples section.

Although immunoassays are primarily described, it should be understoodthat devices described herein may be used for any suitable chemicaland/or biological reaction, and may include, for example, othersolid-phase assays that involve affinity reaction between proteins orother biomolecules (e.g., DNA, RNA, carbohydrates), or non-naturallyoccurring molecules.

Moreover, although many embodiments described herein include the use ofa fluidic connector to connect two channels or two portions of achannel, embodiments herein also include articles and methods forintroducing a sample into a microfluidic system without using a fluidicconnector. For example, in some embodiments, an open-ended fluidicdevice (i.e., a device where only one end is connected to a microfluidicsystem) may be used to introduce a sample into the microfluidic system.

FIG. 6 shows a block diagram 560 of a microfluidic device that iscompatible with using an open-ended device for sample introduction. Themicrofluidic device may contain stored reagents and can be used forperforming an a chemical and/or biological reaction (e.g., animmunoassay). The microfluidic device includes a reagent inlet 562 influid communication with a reagent storage area 564, which may include,for example, one or more channels and/or reservoirs. The device may alsoinclude a sample inlet 565, sample loading area 566, and reaction area568. The reaction area, which may include one or more areas fordetecting a component in a sample, may be in fluid communication withwaste area 570, and may be coupled to outlet 572. In some embodiments,reaction area 568 is an immunoassay area.

FIGS. 7A-7D show an example of a microfluidic system having the featuresdescribed in FIG. 6. Microfluidic system 590 is compatible with anopen-ended fluidic device for introducing a sample into the system. InFIG. 7A, fluid reagents are stored in reagent storage area 564 and dryreagents are stored in reaction area 568. Inlets 562 and 565, and outlet572 are sealed prior to use. As shown in the embodiment illustrated inFIG. 7B, a seal over sample inlet 565 can be pierced, removed, or brokento allow a sample 592 to be introduced into sample inlet 565, which canflow into sample loading area 566, which may include an empty meanderingchannel 594. Flow of the sample may take place initially by capillaryforces. Optionally, a seal may be placed over sample inlet 565 and avacuum can be applied to outlet 572 to cause fluid flow towards theoutlet (FIG. 7C). The sample flows into reaction area 568, followed bythe stored fluid reagents from reagent storage area 564. As shown inFIG. 7D, after all of the reagents have passed through the reactionarea, they may be contained in waste area 570 (or, optionally, may exitout of the device via the outlet).

As described herein, fluids (e.g., samples) can be introduced into amicrofluidic device using a variety of devices such as an open-endedfluidic device and/or a fluidic connector. Although severalconfigurations of such devices are shown in FIGS. 8-13, it should beunderstood that the invention is not limited to these configurations andthat other configurations and/or arrangements are possible.Additionally, although descriptions herein involving sample introductioncomponents (e.g., open-ended fluidic devices and fluidic connectors)primarily describe introduction of samples to microfluidic substrates,such components can be used to introduce any suitable substance such asreagents (e.g., buffers, amplification reagents, a component of atwo-part system), gases, and particles.

For devices used in point-of-care settings, the sample introductioncomponents may be designed to protect the user from occupationalhazards. Additionally, the complexity of the sample-handling step may beminimized to allow the use of the device outside medical laboratories.These factors may be considered when choosing a particular design for asample introduction component.

Sample introduction components such as open-ended fluidic devices andfluidic connectors may include any suitable article having a fluid pathdisposed therein. The sample introduction component (as well as otherchannels of the microfluidic system) may have a consistent or variableinner diameter and may have a length-to-internal diameter ratio of, forexample, greater than 10 to 1, greater than 50 to 1, or greater than 100to 1. Depending upon the application, sample introduction components (ormicrofluidic channels) of any diameter may be used, and in manyapplications it may have an inner diameter of, for example, less than 1cm, less than 5 mm, less than 1 mm, less than 500 microns, less than 200microns, less than 100 microns, or less than 50 microns. A sampleintroduction component (or microfluidic channel) with a greaterlength-to-internal diameter ratio may be useful in visually indicatingthe amount of each fluid contained in the component (or microfluidicchannel). For instance, a linear measurement of a fluid plug in afluidic device or fluidic connector of known inner diameter may give anaccurate indication of the volume or the relative volume of the fluid.In some embodiments, the sample introduction component comprises a tube.Tubes are readily available in different diameters, lengths andmaterials. Tubes may be flexible and may be translucent or transparent.Fluid plugs in a tube may be measured linearly as an indication of thevolume of the plug.

The sample introduction component, if a tube or another shape, mayinclude two or more branches or sections that may be in fluidcommunication with each other and with the remaining interior of thecomponent. In some embodiments, a tube may have two, three, four or morebranches that may be interconnected. The branches and branch junctionsmay or may not include valves. Valves may be used to temporarilysegregate one or more branches, and any liquid contained therein, fromthe remainder of the tube.

In some embodiments, a sample introduction component such as anopen-ended fluidic device or a fluidic connector includes a volumecontrol element. The volume control element can allow a fluid to fill aportion, but not all, of a fluid path of a sample introductioncomponent. The volume control element can be used to meter a particularvolume of fluid for introduction into a microfluidic system. In oneembodiment, a volume control element is a frit, which can be placedinside a fluid path of a sample introduction component to stop furtherfluid from being introduced inside the fluid path after the fluidreaches a particular volume. The volume of fluid (e.g., sample) in thesample introduction component can be defined by the volume of the fluidpath between the entry point (e.g., an inlet) for fluid introduction andthe frit; the remaining volume may be occupied by air.

In another embodiment, a volume control element includes one or moremetering marks that indicate up to which point(s) a fluid should beintroduced into the fluid path. The volume of fluid in the fluid pathmay be controlled by the user.

In yet another embodiment, a volume control element includes a change indiameter (e.g., widening) of a fluid path within the sample introductioncomponent. For instance, an open-ended fluidic device or a fluidicconnector may include a first end (e.g., an opening), a first portion ofa fluid path having a first diameter, a second portion of the fluid pathhaving a second diameter, followed by a second end (e.g., an opening).The second diameter may be greater than the first diameter. The firstdiameter may be favorable for causing fluid to flow into the fluid pathvia capillary forces, while the second diameter may be less favorable(or unsuitable) for capillary action. Accordingly, a fluid may enter thefirst portion of the fluid path via the first end and the fluid may stopentering the fluid path when it reaches the second portion of the fluidpath. In this embodiment, the volume of fluid (e.g., sample) in thesample introduction component can be defined by the volume of the firstportion of the fluid path; the remaining volume (e.g., the secondportion of the fluid path) may be occupied by air. Those of ordinaryskill in the art know how to determine diameters of fluid paths that arefavorable or less favorable for capillary action.

In yet another embodiment, a volume control element includes a patternedsurface within a fluid path of the sample introduction component. Forinstance, a sample introduction component may include a first end (e.g.,an opening), a first portion of a fluid path having a first, hydrophilicsurface, a second portion of the fluid path having a second, hydrophobicsurface, followed by a second end (e.g., an opening). The first,hydrophilic surface can cause a hydrophilic fluid (e.g., an aqueousfluid) to flow into the fluid path via capillary forces, while thesecond, hydrophobic surface is less favorable for capillary action.Accordingly, a fluid may enter the first portion of the fluid path viathe first end and the fluid may stop entering the fluid path when itreaches the second portion of the fluid path. In this embodiment, thevolume of fluid (e.g., sample) in the sample introduction component canbe defined by the volume of the first portion of the fluid path; theremaining volume (e.g., the second portion of the fluid path) may beoccupied by air. In one particular embodiment, a hydrophilic portion ofthe fluid path is defined by the presence of an anti-coagulant (e.g.,heparin, a chelator (e.g., ethylenediamine tetraacetic acid, EDTA) orcitrate), and a hydrophobic portion of the fluid path is defined by theabsence of an anti-coagulant (or the presence of one or more hydrophobicmolecules). Methods and materials for patterning surfaces of fluid pathsare known by those of ordinary skill in the art.

In some embodiments, a sample introduction component such as anopen-ended fluidic device or fluidic connector can include a combinationof volume control elements such as the ones described above. A sampleintroduction component including one or more volume control elements canbe filled using any suitable method such as by capillary forces,application of a vacuum, application of a positive pressure, and by useof valves.

As described in more detail below, sample introduction components can beconnected to a substrate using a variety of methods. For example, asample introduction component and/or substrate may include one or moreof the following: pressure-fittings, friction-fittings, threadedconnectors such as screw fittings, snap fittings, adhesive fittings,clips, magnetic connectors, or other suitable coupling mechanisms.

FIG. 8A shows an example of an open-ended capillary tube 700 (e.g.,open-ended fluidic device) that can be used for introducing a sampleinto an inlet of a device (e.g., sample inlet 565 of FIG. 7A). Tube 700may have an open end 704 (e.g., for inserting into an inlet of adevice); end 702 may either be opened or closed. As shown in FIG. 8B, acapillary tube 710 can also be used as a fluidic connector to connecttwo channels (or portions of a channel) of a microfluidic system, e.g.,as described in connection with FIG. 3. Tube 710 can include opened ends712 and 714. The use of a capillary bent to form a “U”-shape is one ofmany possible devices that can be used to connect two channels (orportions of a channel).

The devices of FIGS. 8A and 8B can be made of any suitable material(e.g., a polymer or ceramic) and may be rigid or flexible. Non-limitingexamples of such materials include glass, quartz, silicon, PTFE(Teflon), polyethylene, polycarbonate, poly(dimethylsiloxane), PMMA,polystyrene, a cyclo-olefin copolymer (COC) and cyclo-olefin polymer(COP). In certain embodiments where the tubes are formed of a flexiblematerial, the tube may be placed in a holder of a sufficiently rigidmaterial to maintain the tube in its final shape. For example, as shownin the embodiment illustrated in FIG. 8C, tube 720 may be positioned ingroove 732 of holder 730 to maintain the shape of the tube. Optionally,a cover 734 may be used to cover the holder and may be attached to theholder, for example, by sealing, gluing, bonding, using adhesives, or bymechanical attachment (e.g., clipping into the holder). In otherembodiments, instead of positioning the tube in a groove, the holder mayinclude raised features (e.g., clips) for securing the tube. Ends 722and 724 may be exposed to allow connection to one or more channels of amicrofluidic system (FIG. 8D).

In another embodiment, an open-ended fluidic device (e.g., a capillarytube) or a portion of a fluidic connector can be made of aradiation-sensitive material such as a flexible plastic that hardensupon exposure to heat or UV light. After folding or bending the devicein the desired shape (e.g., a “U”-shape), exposure to the appropriateradiations can cause the capillary to maintain its new shape.

In yet another embodiment, instead of bending straight capillaries toform a U-shape design, the open-ended fluidic device or fluidicconnector can be manufactured directly in its final form. One exampleincludes a capillary made of glass blown in the curved shape, which canallow sample loading onto the microfluidic device and/or fluidconnection between channels or portions of a channel. Othermanufacturing techniques and materials, including injection molding orextrusion of plastics, can also be used.

As shown in the embodiments illustrated in FIGS. 9A-9F, monolithicdevices 800 and 830 having hollow, elongated volumes (e.g.,microchannels 804) may be used as fluidic connectors. The devices may berigid (e.g., for avoiding the need for the user to bend a capillary) andmay optionally include a handle for simple handling (e.g., a verticalhandle 810 as shown in FIG. 9B or a lateral handle 812 as shown in FIG.9E). In such embodiments, a loop of tubing of an U-shaped capillary canbe replaced by microchannels 804 having any suitable dimensions formedin a substrate 816. The dimensions of the microchannels can be tuned toaccommodate a wide range of volumes of fluid (e.g., 1-1000 μL). Suchdevices can be filled entirely with a fluid (e.g., sample) or may befilled partially with fluid (e.g., using a volume control element tometer the amount of fluid in the fluid path). Moreover, the dimensionsof the microchannels can also be chosen to allow the introduction of thefluid in the channels with capillary forces, or alternatively, the fluidcan be aspirated using vacuum.

The channels may be covered by a cover (e.g., covers 820 and 822), whichmay be, for example, a block, an adhesive film, or a tape. The devicepresented in FIGS. 9A-9C may require a bonding step (e.g., by use of anadhesive) between cover 820 and substrate 816. In some embodiments, sucha bonding step may avoided by applying a cover 822 such as an adhesivefilm (e.g., tape) over the surface of the device (FIGS. 9D-9F).

As illustrated in FIGS. 9A and 9D, devices 800 and 830 may includeaccess ports 806 and 808 (e.g., inlets and outlets) that can allow afluid to be introduced into the fluid path and/or to enable fluidcommunication between channels (or portions of a channel) of amicrofluidic system. The access ports can have any suitable shape toallow formation of a tight seal with the ports of the microfluidicsystem. As shown in the embodiments illustrated in FIG. 9, the ports mayhave a conical shape that are complementary to conical apertures of amicrofluidic device.

In some embodiments, once a fluidic connector is connected to amicrofluidic device (e.g., the devices shown in FIGS. 1, 3, and 4), avacuum is applied to an outlet of the device to cause fluid flow in thesystem. In these embodiments, the vacuum may strengthen the quality ofthe seal between the complementary ports.

Another example of a fluidic connector is shown in FIGS. 10A and 10B. Inthe embodiments illustrated in FIGS. 10A and 10B, fluidic connector 852is prepared by assembling two parts 850. Fluidic connector 852 shows theimplementation of a fluid path 855 within a rigid substrate 858,although in other embodiments, any arbitrary geometry can be usedincluding a meandering channel configuration. The inlet and outlet ports862 and 864 may be part of a conical protrusion 865 to form an air-tightseal with the conical apertures of the microfluidic chip. As describedin more detail below, a more elaborate connection system can beimplemented, such as snapping mechanisms or non-conical fittings. Thefluidic connector can be optimized to allow simple handling for the user(including the addition of a handle to the design), if desired.

In some embodiments described herein, the fluidic connector is connectedto a microfluidic device (e.g., a substrate including microfluidicchannels disposed therein), by inserting the ports of the fluidicconnector in access holes located directly above the microchannel(s) ofthe substrate. As a result, the fluid path of the fluidic connector maybe in a plane orthogonal to the plane of the microchannels of thesubstrate, as shown in FIG. 11A. In some applications, however, thereare advantages to placing the fluidic connector in the same plane as themicrochannel network (e.g., using a lateral connection). One advantageof this configuration may be to maximize the area available forobservation of the microfluidic device (e.g., for highly parallelassays). Another advantage may be to allow stacking of a large numberdevices on top of each other while allowing each device to be accessibleto fluid dispensers or other instruments, which can save storage spacein an instrument. In such embodiments, a fluidic connector 872 may beconnected to an end portion 876 of a substrate 880. In other cases, thefluidic connector may be connected to a substrate at an angle between 90and 180 degrees or between 0 and 90 degrees. Accordingly, fluidicconnectors described herein may be connected to a substrate in anysuitable configuration.

The reliability and simplicity of forming a good (e.g., fluid-tight)seal between a fluidic connector and a microfluidic substrate is acritical design aspect of a device for its use in point-of-caresettings. In that regard, the fluidic connector or the substrate itselfcan include additional features to help the user insert the device ontoor into the microfluidic substrate. For instance, in one embodiment, thefluidic connector includes at least one non-fluidic featurecomplementary to a feature of the substrate so as to form a non-fluidicconnection between the fluidic connector and the substrate uponattachment. The non-fluidic complementary feature may be, for example, aprotruding feature of the fluidic connector and correspondingcomplementary cavities of the microfluidic substrate, which can help theuser align the fluidic connector with the substrate. Moreover, theseguiding features can also help maintain the device in place. In otherinstances, the substrate includes protruding features complementary tocavities of the fluidic connector. In yet another embodiment, a deviceincludes an alignment element associated with the substrate andconstructed and arranged to engage with the fluidic connector andthereby position the connector in a predetermined, set configurationrelative to the substrate. Examples of these and other features aredescribed in more detail below.

FIGS. 12A-12E illustrate embodiments that enable attachment of a fluidicconnector to a microfluidic substrate by snapping the two componentstogether to form a connection. This configuration may be especiallyuseful for applications involving point-of-care diagnostics, since thesnapping mechanism may enable a good seal between the components, andmay decrease the chance of the user mishandling the diagnostic test. Thenoise and/or feel experienced by the user while snapping the fluidicconnector into the substrate can be used as a guide or control forsuccessful attachment of the components.

As illustrated in FIG. 12A, a fluidic connector 900 can include twoidentical first portions 910 (only one is shown) that form a fluid path912 upon closing both halves against each other. In other instances, thefluidic connector includes a single integral piece including a fluidpath 912 disposed therein. End portions 916 and 918 (e.g., an inlet andoutlet) of the fluid path may be connected to a microfluidic substrate(not shown) via features 922 and 924, which may be complementary tofeatures of the substrate. The fluidic connector may also includeopenings 930 for inserting clips 934. The clips may include two or moresnap features (e.g., indentations) 936 and 938; these features may beformed of any suitable material (e.g., a polymer) and may be formed ofthe same or a different material than that of the clip and/or thesubstrate. Feature 938 may be used to connect the clip to first portion910, and feature 936 may be used to connect the clip to the microfluidicsubstrate. Such features may allow the clip to be irreversibly attachedto the fluid connector and/or to the substrate. FIG. 12B illustrates amagnified view of the clip. In other embodiments, the fluidic connectorcan be manufactured with the snap features, which can be directly a partof 910; for example, the fluidic connector may include feature 936without the use of clip 934 (not shown).

As shown in the embodiment illustrated in FIG. 12C, once a clip isinserted into openings 930 (e.g., when feature 938 meets opening 930-B),the clip may be attached to portion 910 of the fluidic connector.Likewise, as illustrated in FIG. 12D, the fluidic connector may beinserted into a portion of a microfluidic substrate 940 to causeattachment of the fluidic connector to the substrate (FIG. 9E). The snapfeatures can guide the fluidic connector to the correct position in themicrofluidic substrate. As described in more detail below, theattachment of a fluidic connector to a substrate may be reversible orirreversible. This attachment can cause fluid communication between afirst channel at position 942 of the substrate and a second channel (ora portion of the first channel) at position 944 of the substrate viafluid path 912. As described herein, the fluidic connector may be loadedwith a sample (e.g., via end portion 916 or 918) before or afterattachment.

As an alternative to the snapping mechanism described in connection withFIGS. 12A-12E, a fluidic connector can be attached to a microfluidicsubstrate using a zip-tie mechanism, as illustrated in FIG. 13. FIG. 13shows a component 955 including features 956 (e.g., protrusions) thatare complementary to portion 960, which includes features 962 (e.g.,indentations). Component 955 may be a part of a fluidic connector andportion 960 may be part of a microfluidic substrate. In some instances,component 955 includes a fluid path 958 disposed therein.

Although features for connecting an article and a substrate, such asthose shown in FIGS. 9, 10, 12 and 13, are described in reference tofluidic connectors and substrates, such features may also be used forconnecting other articles of a device. For instance, such features maybe used for connecting components such as an open-ended fluidic deviceand a substrate, a substrate and a cover, and/or multiple substratelayers of a device.

In embodiments described herein involving an article (e.g., a fluidicconnector) comprising at least one feature complementary to a feature ofthe substrate, the features may be designed to form a reversibleconnection between the article and the substrate. Such embodiments maybe useful, for example, for reusable devices. In other embodiments, suchcomplementary features form an irreversible connection between thearticle and the substrate. The irreversible connection may cause thearticle and the substrate to be integrally connected. As used herein,the term “integrally connected,” when referring to two or more objects,means objects that do not become separated from each other during thecourse of normal use, e.g., cannot be separated manually; separationrequires at least the use of tools, and/or by causing damage to at leastone of the components, for example, by breaking, peeling, or separatingcomponents fastened together via adhesives or tools. Devices includingfeatures forming an irreversible connection may be useful, for example,for one-time-use (e.g., disposable) devices. Such devices may form anirreversible connection so that the user cannot interfere with achemical and/or biological reaction being performed in the device afterconnection.

The examples illustrated in FIGS. 12 and 13 include more than twoconnections (e.g., fluidic or non-fluidic connections) between a fluidicconnector and a microfluidic substrate. This characteristic may beuseful because additional points of connection (e.g., non-fluidicconnections) can increase the stability of the attachment againstmechanical stress (e.g., due to handling by the user) and shocks (e.g.,improper use of the device). Additionally, each additional point ofconnection can increase the area of contact between the fluidicconnector and the substrate, while the area associated with forming thefluid-tight seal between the fluidic connector and the substrate canremain unchanged. Alternatively, a single non-fluidic connection may besufficient to yield good sealing properties.

Although many embodiments described herein include sample introductioncomponents (e.g., fluidic connectors) having a single fluid path, itshould be understood that a sample introduction component may includemore than one fluid path and/or branching fluid paths. For example, asshown in the embodiment illustrated in FIG. 12E, fluidic connector 900may optionally include a secondary flow path 946, which connects inlet947 to flow path 912. This design can allow, for example, theintroduction of a fluid into fluid flow path 912 via inlet 947 andsecondary path 946 after fluidic connector 900 has been connected to asubstrate. Alternatively, a fluid can be introduced into fluid path 912via inlet 947 prior to connection of the fluidic connector andsubstrate.

In addition, sample introduction components such as fluid connectorsdescribed herein may include one or more sampling elements used toreceive a fluid sample from a biological entity. The sampling elementmay be in the form of a needle or swab, for example. The samplingelement may be reversibly or irreversibly attached to a sampleintroduction component. In some instances, the sampling element canpuncture a biological component. For instance, as shown in theembodiment illustrated in FIG. 12E, fluidic connector 900 may include a(sterilized) sampling element 948, e.g., in the form of a hollow, sharppoint (e.g., a needle), that may be used to puncture a component such ashuman skin. This configuration can allow the sampling element to receivea fluid sample from the biological component and can enable transfer ofa fluid from the biological entity to fluid path 912 (e.g., by capillaryforces). After fluid has been introduced into inlet 947, secondary fluidpath 946 can be blocked, e.g., using component 949, which may have ashape complementary to that of fluid path 946. This blocking can preventfluid from re-entering the secondary fluid path such that there is onlyone fluid path for flow. This arrangement can also prevent the user frombeing exposed further to sampling element 948.

In another embodiment, component 949 (optionally including a fluid path)can be used to obtain a sample, and upon insertion of the component intosecondary fluid path 946, the sample can be transferred from thecomponent to fluid path 912. In certain embodiments, insertion of thecomponent prevents fluid from re-entering the secondary fluid path suchthat there is only one fluid path for flow.

In some embodiments, a sample introduction component includes a samplingelement connected directly to a primary fluid path. For instance, in theembodiment illustrated in FIG. 10, conical protrusions 865, which may becomplementary to a feature of a microfluidic substrate, may includesampling elements at the ends that can allow puncture of a biologicalcomponent. Sampling elements may also be present as part of anopen-ended fluidic device (e.g., as shown in FIG. 8A) and/or otherfluidic connectors described herein (e.g., FIG. 8B).

Devices described herein may optionally include an alignment elementassociated with the substrate. The alignment element may be constructedand arranged to engage with the fluidic connector and thereby positionthe fluidic connector in a predetermined, set configuration relative tothe substrate. As shown in the embodiments illustrated in FIGS. 14A and14B, device 964 may include a substrate 966, a fluidic connector 968,and an alignment element 980. Substrate 966 may include a microfluidicsystem such as one described herein, e.g., as shown in FIGS. 1-7, 11,17-18. The microfluidic system may comprise, for example, at least afirst microfluidic channel including an inlet and an outlet and a secondmicrofluidic channel including an inlet and an outlet (not shown).Fluidic connector 968, which may have a configuration as describedherein and may be constructed for matching connection to the substrate.The fluidic connector may include a fluid path 970 having a fluid pathinlet 972 and a fluid path outlet 974. Upon connection of the fluidicconnector to the substrate, the fluid path inlet may connect to theoutlet of the first microfluidic channel of the substrate, and the fluidpath outlet 974 may connect to the inlet of the second microfluidicchannel of the substrate. This connection can result in fluidcommunication between the first and second microfluidic channels of thesubstrate.

As shown in the illustrative embodiments of FIGS. 14A and 14B, thedevice may include an alignment element 980 associated with thesubstrate and extending approximately perpendicular to the substrate.For example, while substrate 966 (as well as the first and secondmicrofluidic channels) lies generally in the plane defined betweenarrows 975 and 977, alignment element 980 extends generallyperpendicular to the substrate in the plane defined by arrows 975 and976. In other embodiments, the alignment element may extendapproximately parallel to the substrate.

As illustrated, alignment element 980 includes a cavity 981 constructedand arranged to receive and engage the fluidic connector and therebyposition the connector in a predetermined, set configuration relative tothe substrate. The cavity may have a depth of, for example, at least 0.5cm, at least 1 cm, at least 1.5 cm, at least 2 cm, or at least 3 cm(e.g., as measured from the position of the fluid path inlet and/orfluid path outlet upon engagement of the fluidic connector and thealignment element). The cavity may have a depth similar or equal to theheight of the fluidic connector. The cavity does not necessarily have toencompass all sides of the fluidic connector, as long as it isconstructed and arranged to receive and engage the fluidic connector andthereby position the connector in a predetermined, set configurationrelative to the substrate.

In some embodiments, the configuration of the alignment element and thefluidic connector may be adapted to allow insertion of the fluidicconnector into the alignment element by a sliding motion. For example,the fluidic connector may slide against one or more surfaces of thealignment element when the fluidic connector is inserted into thealignment element.

An alignment element may have any suitable configuration for engaging afluid connector. In some embodiments, the alignment element (or a cavityof an alignment element) may be in contact with 1, 2, 3, 4 or moresurfaces, e.g., surfaces 984, 985, 986, and/or 987, of the fluidconnector upon engagement. One or more surfaces of the alignment elementin contact with the fluidic connector may extend from the substrate,e.g., along the planes defined between arrows 976 and 977, betweenarrows 976 and 975, and therebetween.

In addition, all or portion of the alignment element may have a height,thickness, or a depth (e.g., for insertion of a fluidic connector) thatmay be, for example, greater than or equal to at least 1, 2, 3, 4, 5,etc., times the thickness 979 of the substrate. The alignment elementmay have a height or thickness of, for example, at least 0.5 cm, atleast 1 cm, at least 1.5 cm, at least 2 cm, or at least 3 cm (e.g., asmeasured from the position of the fluid path inlet and/or fluid pathoutlet upon engagement of the fluidic connector and the alignmentelement). A larger height/thickness of the alignment element may allow,in some embodiments, further stabilization and/or guidance of thefluidic connector into the alignment element. The dimensions can varyand may depend on a variety of factors such as the dimensions of thefluidic connector and the substrate.

Optionally, the alignment element may include one or more engagingcomponents that may engage a portion of the fluidic connector. FIG. 14Ashows the fluidic connector having engaging components 982. The engagingcomponent may have a height of, for example, at least 0.5 cm, at least 1cm, at least 1.5 cm, or at least 2 cm as measured from the position ofthe fluid path inlet and/or fluid path outlet upon engagement of thefluidic connector and the alignment element.

In some cases, the alignment element includes an engaging componentcomplimentary to an engaging component of the fluidic connector. Anengaging component may include, for example, a groove or otherindentation, a protrusion (e.g., as shown in FIG. 13), and/or amechanism such as o-ring that may be at least partially deformable. Itshould be understood that an engaging component may have any suitableshape and/or form. In some cases, an engaging component creates asubstantial resistance to movement of the fluidic connector relative tothe substrate and/or alignment element upon the alignment elementreceiving the fluidic component (e.g., upon insertion of the fluidiccomponent into the alignment element) and/or during intended use of thedevice. For example, the single act of inserting fluidic connector 968into the cavity of alignment element 980 may cause the engagingcomponents of the fluidic connector and alignment element to interact,thereby creating a substantial resistance to movement of the fluidicconnector relative to the substrate and/or alignment element. Therefore,in certain embodiments, separate clamps or other fastening mechanisms,and/or secondary steps for fastening, are not required.

In some embodiments, the engaging component causes the fluidic connectorto be integrally connected to the alignment element. In one particularembodiment, engaging components are snap features that may clip into afeature of the alignment element (or fluidic connector). Such and otherfeatures can allow, in some embodiments, the fluidic connector to beirreversibly attached (e.g., integrally connected) to the alignmentelement and/or to the substrate. In other cases, the alignment elementand the fluidic connector are designed to be reversibly attached to oneanother. Accordingly, an engagement component may facilitate theengagement of the fluidic connector and the substrate in apredetermined, set configuration relative to the substrate upon theirconnection.

In some embodiments, the configuration of a cavity and/or an engagingsurface of an alignment element causes the fluid path of the fluidconnector to lie approximately perpendicular to the substrate (and,therefore, approximately perpendicular to the microfluidic channelswithin the substrate). For example, as illustrated in FIGS. 14A and 14B,fluid path 970 is approximately perpendicular to the substrate in theplane defined by arrows 975 and 976. In other embodiments, a fluid pathof the fluid connector lies at an angle between 90 and 180 degrees orbetween 0 and 90 degrees relative to the substrate.

Although FIGS. 14A and 14B show alignment element 980 positioned at oneend of the substrate, in other embodiments, an alignment component canextend along the length, L, of the substrate, e.g., towards opposingends of the substrate. For example, the alignment component may be ablock having a length and width similar to that of the substrate, butmay include a cavity where the fluidic connector is to be inserted.Furthermore, although FIGS. 14A and 14B show alignment element 980 inthe form of two components, in some embodiments an alignment element maybe in the form of a single component. In other embodiments, thealignment element is in the form of more than two components.

In some embodiments, the alignment element and the substrate are in theform of a single piece of material, which, in some cases, can befabricated in one step, e.g., by injection molding. For example, asshown in the exemplary embodiment of FIGS. 15A and 15B, alignmentelement 990 may be part of a substrate 991 containing a microfluidicsystem.

In contrast, as shown in the exemplary embodiment of FIGS. 16A and 16B,a substrate 992, which includes a microfluidic system, and an alignmentelement 994 are separate parts that can be joined together prior to use.The alignment element and the substrate may be connected by insertingclips 934 into portions of the substrate, e.g., as described inconnection with FIGS. 12A and 12B. This connection may be performedprior the user receiver using the device. In other instances, the usercan insert the alignment element into the substrate, followed by thefluidic connector into the alignment element. Alternatively, the usermay insert the fluidic connector into the alignment element, followed bythe alignment element into the substrate.

FIGS. 14 and 15 also show an alignment element including engagingcomponents 983, which engage with engaging components 982 of the fluidicconnector. The device may be configured such that a fluidic connector969 can be inserted into the alignment element in the direction of arrow978, while preventing or inhibiting removal of the fluidic componentfrom the alignment element after insertion (e.g., in the oppositedirection of arrow 978).

It should be understood that alignment elements may be combined withother features described herein. For example, an alignment element maybe associated with a fluidic connector that includes at least onenon-fluidic feature complementary to a feature of the substrate so as toform a non-fluidic connection between the fluidic connector and thesubstrate upon attachment, e.g., as described in connection with FIGS. 1and 12.

There are several advantages of using microfluidic devices with fluidicconnectors, especially when performing chemical and/or biologicalreactions (e.g., immunoassays) in the device. Accordingly, devicesdescribed herein may have one or more advantages such as: (a) use ofsmall amounts of sample with little or no sample waste, (b) long-termstability of chemical and/or biological reagents stored in the device,(c) reduction of cross-contamination between stored reagents and/orbetween sample and reagent, (d) sample metering, (e) ease of use tountrained users for introducing a sample into the device, (f) efficientmixing of reagents, and (g) assay reliability. In some embodiments, thedevices have all of the advantages listed above.

Small amounts of sample can be used with little or no sample wastebecause fluidic connectors (as well as open-ended fluidic devices) canbe designed to have an internal volume matching the volume of samplerequired for performing the chemical and/or biological reaction. Thiscan reduce the amount of dead volume in a system. Optionally, asdescribed above, fluidic connectors and open-ended fluidic devices caninclude one or more volume control elements to allow collection of aparticular volume of sample.

Devices described herein may be used for point-of-care applications, andcan be manufactured several months (or years) prior to first use. Insome embodiments requiring storage of components in the device prior tofirst use, it is important that all biomolecules and reagents introducedat the time of manufacturing remain stable for extended periods of time.For example, in a reaction area, capture antibodies can be physisorbedto the surface of the microchannels, and can be stabilized in a dry formusing stabilizers (e.g., trehalose).

It has been demonstrated previously that the storage of the reagents inthe form of liquid plugs separated by air gaps were stable for extendedperiods of time (see, for example, International Patent Publication No.WO2005/072858 (International Patent Application Serial No.PCT/US2005/003514), filed Jan. 26, 2005 and entitled “Fluid DeliverySystem and Method,” which his incorporated herein by reference in itsentirety).

Both liquid and dry reagents may be stored on a single microfluidicsubstrate. As described herein, in some embodiments, a channelcontaining a liquid reagent is not in fluid communication with a channelcontaining a dry reagent since, depending on the particularenvironmental (e.g., storage) conditions, if the channels containing thereagents are in fluid communication with one another, transport of watervapors can result in the wet reagent drying out and dry molecules beinghydrated. This can affect the long-term stability of all reagents storedon certain devices. A system involving the use of a fluidic connectorand a microfluidic substrate including dry reagents physically separated(e.g., in different channels) and not in fluidic communication with thewet reagents can allow fluid communication only at the time of use ofthe microfluidic device. This configuration can enhance the stability ofthe reagents for long-term storage. In other embodiments, however,liquid and dry reagents can be stored in fluid communication with oneanother (e.g., for shorter-term storage).

Another advantage of microfluidic devices described herein may bereduction of cross-contamination between stored reagents and/or betweensample and reagent. Cross contamination may occur, in certainembodiments, at intersections between microfluidic channels, where plugsof reagents can get caught. These reagents can contaminate subsequentreagents flowing past the same intersection. The use of a fluidicconnector can greatly simplify a microchannel network, reducing orobviating the number of intersection(s) on a device, and thus anypotential cross-contamination problems.

Sample metering is another important requirement for many microfluidicapplications. Often this is performed off-chip and an accurate samplevolume is loaded onto the chip with the hope that the entire volume willflow inside the device. With fluidic connectors described herein, thevolume of sample that can be introduced inside the microfluidic devicecan be accurately measured, and the entire volume of sample can be sentto a reaction area of the device.

As described herein, several designs of sample introduction components(e.g., fluidic connectors and open-ended fluidic devices) can be used byuntrained users (see, for example, the embodiments described inconnection with FIGS. 8-16). These components can be designed tofacilitate the sample loading procedure and to allow simple attachmentof a fluidic connector to a microfluidic substrate. Such devices may beespecially useful in point-of-care settings by untrained users.

Another advantage of systems and methods described herein may includeefficient mixing of reagents on a device. An example of efficient mixinghas been described herein in connection with silver enhancementchemistry based on the reduction of silver ions by a reducing agent(e.g., hydroquinone) by a catalyst (e.g., a noble metal). In embodimentsinvolving immunoassays, secondary antibodies can be labeled with goldcolloids (catalyst). In the presence of a mixture of silver ions andhydroquinone, multiple layers of silver can be created at the surface ofthe gold colloid, increasing the size of the colloid. After about 10minutes of amplification, the size of the colloid can increase by afactor of, for example, about 1000, yielding on the surface grains ofsilver that can be observed with an optical setup. To achieve goodamplification results (e.g., a large signal amplification with littleamplification of background), the amplification reagent can be storedseparately, e.g., in separated channels or containers, and mixed onlyimmediately before use. In microfluidic devices, the cross-sectionaldimensions of the channel may be small and flows may be laminar, meaningmixing occurs primarily by diffusion, which is typically inefficient andslow. However, the laminar character of the flow of reagents may bedecreased when traveling through a fluid path of a fluid connector,since the fluid path may have a relatively larger cross-sectionaldimension (and, therefore, a relatively larger volume) than the that ofthe microchannels of the substrate. Accordingly, in certain embodiments,each fluid connector can act as a chaotic mixer and can significantlyimprove the mixing of two or more reagents. In the example describedabove, this mixing can improve the reproducibility of the amplificationchemistry.

In some embodiments described herein, microfluidic devices include onlya single interconnected channel with, for example, less than 5, 4, 3, 2,or 1 channel intersection(s) when in use (e.g., upon attachment of afluidic connector and a substrate). A layout based on a single channelwith minimal or no intersections may be reliable because there is onlyone possible flow path for any fluid to travel across the microfluidicchip. In these configurations, the reliability of a chemical and/orbiological reaction to be performed in the device is greatly improvedcompared to designs having many intersections. This improvement occursbecause at each intersection (e.g., a 3-way intersection or more), thefluid has the potential to enter the wrong channel. The ability to loada sample without channel intersections can eliminate risk of fluidentering the wrong channel. Because an intersection may represent a riskfactor that must be taken into account in product development, controls(either on-chip or based on external inspection) must be set up toinsure correct fluid behavior at each interconnection. In certainembodiments described herein, the need for such additional controls canbe alleviated.

As described above, reagents can be stored in a microfluidic deviceusing a variety of methods. Such methods may depend at least in part onthe form in which the reagent is stored (e.g., dried or wet), theconfiguration of the channels within microfluidic system (e.g., whetherthe channels are interconnected or unconnected), the length of time ofstorage, and/or the particular application.

Referring back to FIG. 2, in some embodiments, a first reagent (orseries of reagents) is positioned in a first channel formed in asubstrate, such as in a channel or reservoir of reagent storage area 64.A second reagent (or series of reagents) may be positioned in a secondchannel formed in a substrate, such as a channel or reservoir ofimmunoassay area 68. In some cases, the first and second channels arenot in fluid communication with one another during the positioning ofthe reagents. The first and/or second reagent may be positioned in theirrespective channels by first flowing the reagents in the channels andthen sealing any inlet(s) and/or outlet(s) of the channels.

The first and/or second reagents may be substantially altered afterbeing positioned in their respective channels. For instance, in somecases the first and/or second reagents is dried after flowing thereagent(s) in a channel. Optionally, the dried reagents may be treatedwith a third reagent (e.g., a blocking agent) which may, for example,reduce non-specific adsorption during carrying out of an assay. Thedried reagent(s) may be stored in a channel by sealing one or moreinlets and/or outlets of the microfluidic channel.

In some instances, a reagent is positioned in a channel prior tocomplete fabrication of a microfluidic channel system. A microfluidicchannel system is not complete if, for example, a system that isdesigned to have enclosed channels has channels that are not yetcompletely enclosed. A channel is enclosed if at least one portion ofthe channel has a cross-section that is completely enclosed, or if theentire channel is completely enclosed along its entire length with theexception of its inlet(s) and/or outlet(s).

In some embodiments, one or more reagents is positioned on a detectionzone of a substrate by placing a droplet of the reagent at the detectionzone (e.g., detection zones 162, 164, 166, and 168 of FIG. 3). Thesubstrate may be formed of a hydrophobic material, which can preventspreading of aqueous reagents across adjacent detection zones. Thereagents at the detection zones may be dried and a cover may be placedadjacent the substrate to complete fabrication of the channel system.Subsequently, any inlet(s) and/or outlet(s) of the channel can besealed.

In another embodiment, one or more reagents is positioned (e.g.,patterned) on a cover, and then the cover is used to enclose amicrofluidic channel system formed in a substrate. The reagents on thecover may be aligned with certain areas within the microfluidic system.For instance, in one particular embodiment, reagents (e.g., antibodies)are patterned in an arrangement (e.g., shape and dimension) that ismatched with detection zones 162, 164, 166, and 168 of FIG. 3. Thereagents can be dried, and then the cover can be sealed against thesubstrate such that the reagents are positioned in the detection zonesof the microfluidic system. The cover can be, for example, abiocompatible adhesive (e.g., prepared on a substrate) and can be madeof a polymer (e.g., PE, COC, PVC) or an inorganic material. For someapplications, the material and dimensions of a cover are chosen suchthat the cover is substantially impermeable to water vapor. In otherembodiments, the cover can be non-adhesive, but may bond thermally tothe microfluidic substrate by direct application of heat, laser energy,or ultrasonic energy. Any inlet(s) and/or outlet(s) of the channel canbe sealed (e.g., by placing an adhesive over the inlet(s) and/oroutlet(s)) after introducing reagents into the device.

Wet reagents are typically stored in a microfluidic system afterchannels of the system have been completely covered. A fluid reagent tobe stored in the system may be introduced into an inlet of a channel,and after at least partially filling the channel with the fluid, theinlet(s) and/or outlet(s) of the channel can be sealed, for example, toretain the fluid and to prevent contamination from external sources.

In some instances, one or more fluids to be stored in a microfluidicsystem is transferred from a vessel (e.g., a cartridge, tube, or fluidicconnector) to the microfluidic system. The vessel may contain, forexample, two or more distinct fluids separated by a third fluid that isimmiscible with both. Any number of distinct fluids may be contained ina vessel. For example, in one embodiment, the vessel is a tube thatincludes a reagent solution plug followed by an air plug, followed by arinse solution plug. An additional air plug may separate the first rinsesolution plug from a second rinse solution plug. The liquid plugs mayretain their relative positions in the tube and may be prevented fromcontacting each other by the interspaced air plugs. Articles and methodsfor delivering fluids to a microfluidic system are described in moredetail in International Patent Publication No. WO2005/072858(International Patent Application Serial No. PCT/US2005/003514), filedJan. 26, 2005 and entitled “Fluid Delivery System and Method,” which hisincorporated herein by reference in its entirety.

Using a vessel containing fluid plugs in linear order can allowintroduction of fluid from the vessel to a microfluidic system in aparticular sequence. These fluids can then be stored in the particularsequence in the microfluidic system (e.g., in a reagent storage area).The inlet(s) and/or outlet(s) of the channel containing the fluids canbe sealed, for example, to retain the fluid and to prevent contaminationfrom external sources. In some embodiments, a particular sequence offluids is contained in a fluidic connector. For example, the particularsequence of fluids may include reagents (e.g., a sample, a buffer, acomponent that binds with the sample, etc.) positioned in series and maybe optionally separated by immiscible fluids. This sequence of fluidscan be introduced into a microfluidic substrate by fluidly connectingthe fluidic connector and the substrate.

In some embodiments, a microfluidic channel or fluidic connector havinga relatively large length-to-internal diameter ratio (or high surfacearea to volume ratio) is used to store one or more fluids. Thisconfiguration can allow a linear measurement of one or more fluid plugsin a fluidic device or fluidic connector of known inner diameter, andmay give an accurate indication of the volume or the relative volume ofthe fluid. This feature may be useful for determining if an accurate orcorrect amount of fluid is contained in a channel, especially afterlong-term or short-term storage of one or more fluids in the channel.For example, if the channel has a relatively large length-to-internaldiameter ratio (e.g., greater than 10 to 1, greater than 50 to 1, orgreater than 100 to 1), a user may be able to determine if an accurateor correct amount of fluid is contained in the channel by simpleinspection, since the loss of fluid (e.g., by evaporation) can result inair bubbles or the presence of empty portions in the channel. If suchair bubbles or empty portions are present, or the amount of fluid in thechannel it outside of a range indicated on the device, the user may bewarned (e.g., by instructions that accompany the device) that the deviceshould not be used. This visual inspection may be difficult in certaindevices that use reservoirs having relatively a small length-to-internaldiameter ratio (or a low surface area to volume ratio) for storingfluids.

Reagents can be stored in a microfluidic system for various amounts oftime. For example, a reagent may be stored for longer than 1 hour,longer than 6 hours, longer than 12 hours, longer than 1 day, longerthan 1 week, longer than 1 month, longer than 3 months, longer than 6months, longer than 1 year, or longer than 2 years. Optionally, themicrofluidic system may be treated in a suitable manner in order toprolong storage. For instance, microfluidic systems having storedreagents contained therein may be vacuum sealed, stored in a darkenvironment, and/or stored at low temperatures (e.g., below 0 degreesC.). The length of storage depends on one or more factors such as theparticular reagents used, the form of the stored reagents (e.g., wet ordry), the dimensions and materials used to form the substrate and coverlayer(s), the method of adhering the substrate and cover layer(s), andhow the device is treated or stored as a whole.

As described herein, different sections of a microfluidic channel orreservoir, especially within a reaction area, can be each modified witha different species (e.g., capture molecule) that can be stored in thechannel or reservoir, so that a sample traveling throughout themicrochannel channel can travel successively over each of the species.The sections of the microfluidic channel may be, for example, detectionzones (e.g., meandering channel regions) as described herein inconnection with FIGS. 2-7 and 14-17. In some embodiments, these sectionsare connected in series. In other embodiments, the sections areconnected in parallel. In yet other embodiments, a device may include acombination of sections connected in series and parallel. In embodimentsincluding detection zones connected in series (and/or in parallel),multiple components of the sample can be tested individually in each ofthe detection zones of the channel. The detection zones may havedifferent configurations depending on the application; for example, adetection zone may be in the form of a reservoir (which may be supportedby an array of pillars) or a meandering channel region, as described infurther detail below. In certain embodiments, a device includes aplurality (e.g., at least 2, 4, 6, 8, 10, or more) of sections, eachsection comprising a single chemical and/or biological species that canundergo a chemical and/or biological reaction (or which may beunreactive towards particular components of a sample, as in a negativecontrol). The chemical and/or biological species in one section may bethe same (e.g., same species and concentration) or different (e.g.,different species and/or concentration) as the species of anothersection.

To simplify signal quantification, each detection zone (e.g., meanderingchannel region) may have a relatively large area compared to across-sectional dimension of a microfluidic channel of the system. Forexample, the detection zone may have an area of greater than 0.1 mm²,greater than 0.2 mm², greater than 0.4 mm², greater than 0.6 mm²,greater than 0.8 mm², or greater than 1 cm². The area may be, forexample, between 0.1 mm² to 0.3 mm², between 0.2 mm² to 0.4 mm², between0.4 mm² to 0.6 mm², or between 0.5 mm² to 1 cm². Different proportionsof the detection zone may comprise an optical detection pathway. Forexample, at least 20%, at least 40%, at least 50%, at least 60%, or atleast 80% of the area of the detection zone may comprise an opticaldetection pathway. The area spanned by the detection zone may be definedby the rectangular area bound by outermost points of the detection zonealong each axis. A signal produced in the detection zone may behomogeneously spread over a large area, thus simplifying the alignmentof an optical readout device.

As shown in the embodiments illustrated in FIGS. 17A-17C, device 1000may include a reaction area 1010 having several detection zones 1012,1014, 1016, and 1018. Each of these detection zones may be in the formof a meandering region 1012-A, 1014-A, 1016-A, and 1018-A, respectively(FIG. 14B). The meandering regions include several channel segments1024. The meandering regions can be connected to one another (i.e., influid communication with one another) via microfluidic channel 1020.Fluid flowing in channel 1020, e.g., in the direction of arrow 1028, canflow through the meandering regions sequentially.

As described herein, a surface of the meandering channel in eachmeandering region can be modified with one or more biomolecules (e.g.,in the form of a stored reagent) for a particular application. Toprovide on-chip quality control, meandering region 1018-A can bemodified with a blocking solution such as BSA or Tween 20 to provide anegative reference for the assay. In a similar fashion, meanderingregion 1012-A can be modified with a positive control. The choice ofthese standards may be such that after successful assay completion, thenegative standard should indicate no signal (or very weak backgroundsignal), and the positive signal should indicate a clear signal. Ingeneral, the choice of the reagent/biomolecule to be immobilized in eachmeandering region can be governed by the particular test to beperformed; for example, for the measurement of total human IgG in serum,anti-human antibodies can be physisorbed in meandering regions 1014-Aand 1016-A.

FIG. 17C is a schematic diagram showing the meandering regions afterperforming a chemical and/or biological reaction in the meanderingregions. Meandering region 1018-B used as a negative control has a weaksignal and appears light grey. Meandering regions 1014-B and 1016-B thatincluded physisorbed reagents that can be used for determining acomponent in the sample may include a detectable signal (e.g., a grayfilm). Meandering region 1018-B used as a positive control may include astrong signal (e.g., a black film).

FIGS. 17A-17C show an example of a multiplex assay that can be performedin a microfluidic device described herein. In other embodiments,additional meandering regions (e.g., greater than 5, 8, 10, 15, or 20meandering regions, which may be connected in series and/or parallel),can be included on a device to allow detection of additional componentsin a sample.

After performing a chemical and/or biological reaction in a detectionzone (e.g., meandering region), a signal may appear in the detectionzone. The type and strength of the signal may depend on the choice oflabel and/or amplification chemistry used. For example, in oneembodiment, silver enhancement chemistry can be used to produce a signalthat can be detected by a simple detector, such as the one described inInternational Patent Publication No. WO2005/066613 (International PatentApplication Serial No. PCT/US2004/043585), filed Dec. 20, 2004 andentitled “Assay Device and Method”, which is incorporated herein byreference in its entirety.

When more than one chemical and/or biological reaction (e.g., amultiplex assay) is performed on a device, the signal acquisition can becarried out by moving a detector over each detection zone. In analternative approach, a single detector can detect signal(s) in each ofthe detection zones simultaneously. In another embodiment, an analyzercan include, for example, a number of parallel opticalsensors/detectors, each aligned with a detection zone and connected tothe electronics of a reader (e.g., FIGS. 18A and 18B). FIGS. 18A and 18Billustrate an optical system 1050 at rest (FIG. 18A) and duringmeasurement (FIG. 18B). As shown in the embodiment illustrated in FIG.18A, optical system 1050 includes a device 1054 having a detection area1060 including detection zones 1062, 1064, and 1066. The optical setupalso includes an article 1070 comprising an array of light sources 1072,1074, and 1076, as well as an article 1080 comprising an array ofdetectors 1082, 1084, and 1086. In some embodiments, articles 1070 and1080 are combined to form an analyzer. The light sources and detectorsmay be aligned with the detection zones of the device. Duringmeasurement, an optical pathway 1092 between optical light source 1072,detection zone 1062, and detector 1082 allows determination of a signalin the detection zone. Parallel optical pathways 1094 and 1096 can allowsimultaneous determination of signals in detection zones 1064 and 1066,respectively.

The interior of an analyzer can be designed to allow simultaneousreading (e.g., detection or determination of a signal) in all detectionzones without interference between each optical pathway in the system.For example, in the embodiment illustrated in FIG. 19, system 1100includes light source 1072 and detector 1082 aligned with each other anddetection zone 1062. Additionally, light source 1074 can be aligned withdetection zone 1064 and detector 1084 and light source 1076 can bealigned with detection zone 1066 and detector 1086. The light sourcesand detectors may be in electronic communication with control unit 1098(e.g., a microprocessor). In some embodiments, one or more opticalfilters can be positioned between a detector and a detection zone.Additionally and/or alternatively, each detector may include anelectronic filter for filtering different wavelengths of light. Tofurther reduce cross-talk between optical pathways, the light from eachlight source can be modulated at a frequency different for each opticalpathway; that is, optical pathways 1092, 1094, and 1096 may each includelight of different wavelengths. The electronic signal generated by lightsource 1072 can be differentiated from noise signal arising from byneighboring light sources 1074 and 1076 by using, for example, anelectronic filter. In a different approach, the readout can be performedsequentially to avoid noise signal(s) arising from the neighboring lightsources. Using a light source-detector pair for each detection zone maybe advantageous when the optical components are relatively simple and/orinexpensive.

In some embodiments, one or more optical components can be sharedbetween detection zones. For instance, in the embodiment illustrated inFIG. 20, a system 1120 includes a detector 1072 and an optical element1122 (e.g., a collecting optic such as an optical fiber), which arealigned with each other and with detection zone 1062. Similarly, thesystem includes a detector 1074 and an optical element 1124 aligned withdetection zone 1064, as well as a detector 1076 and an optical element1126 aligned with detection zone 1066. The optical elements may all beconnected to an optical switch 1130 and to a common light detector 1132,such as an avalanche photodiode or a photomultiplier tube. The commondetector may be used to detect signals in each of the detection zones(e.g., sequentially). The light from each detection zone can becollected by the optical elements, which can be aligned underneath eachdetection zone.

A variety of determination (e.g., measuring, quantifying, detecting, andqualifying) techniques may be used. Determination techniques may includeoptically-based techniques such as light transmission, light absorbance,light scattering, light reflection and visual techniques. Determinationtechniques may also include luminescence techniques such asphotoluminescence (e.g., fluorescence), chemiluminescence,bioluminescence, and/or electrochemiluminescence. Those of ordinaryskill in the art know how to modify microfluidic devices in accordancewith the determination technique used. For instance, for devicesincluding chemiluminescent species used for determination, an opaqueand/or dark background may be preferred. For determination using metalcolloids, a transparent background may be preferred. Furthermore, anysuitable detector may be used with devices described herein. Forexample, simplified optical detectors, as well as conventionalspectrophotometers and optical readers (e.g., 96-well plate readers) canbe used.

In some embodiments, determination techniques may measure conductivity.For example, microelectrodes placed at opposite ends of a portion of amicrofluidic channel may be used to measure the deposition of aconductive material, for example an electrolessly deposited metal. As agreater number of individual particles of metal grow and contact eachother, conductivity may increase and provide an indication of the amountof conductor material, e.g., metal, that has been deposited on theportion. Therefore, conductivity or resistance may be used as aquantitative measure of analyte concentration.

Another analytical technique may include measuring a changingconcentration of a precursor from the time the precursor enters themicrofluidic channel until the time the precursor exits the channel. Forexample, if a silver salt solution is used (e.g., nitrate, lactate,citrate or acetate), a silver-sensitive electrode may be capable ofmeasuring a loss in silver concentration due to the deposition of silverin a channel as the precursor passes through the channel.

Different optical detection techniques provide a number of options fordetermining reaction (e.g., assay) results. In some embodiments, themeasurement of transmission or absorbance means that light can bedetected at the same wavelength at which it is emitted from a lightsource. Although the light source can be a narrow band source emittingat a single wavelength it may also may be a broad spectrum source,emitting over a range of wavelengths, as many opaque materials caneffectively block a wide range of wavelengths. The system may beoperated with a minimum of optical devices (e.g., a simplified opticaldetector). For instance, the determining device may be free of aphotomultiplier, may be free of a wavelength selector such as a grating,prism or filter, may be free of a device to direct or columnate lightsuch as a columnator, or may be free of magnifying optics (e.g.,lenses). Elimination or reduction of these features can result in a lessexpensive, more robust device.

In one embodiment, the light source can be pulse modulated, for example,at a frequency of 1,000 Hz. To match the pulse modulated light source, adetector may include a filter operating at the same frequency. By usinga pulse modulated light source it has been found that the system can beless sensitive to extrinsic sources of light. Therefore, an assay mayrun under various light conditions, including broad daylight, that mightmake it impractical to use existing techniques. Experimental resultsindicate that by using a pulse modulated light source and filter,results are consistent regardless of the light conditions under whichthe test is run.

The light source may be a LED (light-emitting diode) or a laser diode.For example, an InGaAlP red semiconductor laser diode emitting at 654 nmmay be used. The photodetector may be any device capable of detectingthe transmission of light that is emitted by the light source. One typeof photodetector is an optical integrated circuit (IC) including aphotodiode having a peak sensitivity at 700 nm, an amplifier and avoltage regulator. If the light source is pulse modulated, thephotodetector may include a filter to remove the effect of light that isnot at the selected frequency. When multiple and neighboring signals aredetected at the same time, the light source used for each detection zonecan be modulated at a frequency sufficiently different from that of itsneighboring light source. In this configuration, the detector can beassorted with a filter of matching fervency (compared to its attributedlight source), thereby avoiding interfering light form neighboringoptical pairs.

As described herein, a meandering channel of a reaction area may beconfigured and arranged to align with a detector such that uponalignment, the detector can measure a single signal through more thanone adjacent segment of the meandering channel. In some embodiments, thedetector is able to detect a signal within at least a portion of thearea of the meandering channel and through more than one segment of themeandering channel such that a first portion of the signal, measuredfrom a first segment of the meandering channel, is similar to a secondportion of the signal, measured from a second segment of the meanderingchannel. In such embodiments, because the signal is present as a part ofmore than one segment of the meandering channel, there is no need forprecise alignment between a detector and a detection zone.

The positioning of the detector over the detection zone (e.g., ameandering region) without the need for precision is an advantage, sinceexternal (and possibly, expensive) equipment such as microscopes,lenses, and alignment stages are not required (although they may be usedin certain embodiments). Instead, alignment can be performed by eye, orby low-cost methods that do not require an alignment step by the user.In one embodiment, a device comprising a meandering region can be placedin a simple holder (e.g., in a cavity having the same shape as thedevice), and the measurement area can be automatically located in a beamof light of the detector. Possible causes of misalignment caused by, forinstance, chip-to-chip variations, the exact location of the chip in theholder, and normal usage of the device, are negligible compared to thedimensions of the measurement area. As a result, the meandering regioncan stay within the beam of light and detection is not interrupted dueto these variations.

The detector may detect a signal within all, or a portion, of adetection zone (e.g., including a meandering region). In other words,different amounts of the meandering region may be used as an opticaldetection pathway. For instance, the detector may detect a signal withinat least 15% of the detection zone, at least 20% of the detection zone,at least 25% of the detection zone, within at least 50% of the detectionzone, or within at least 75% of the detection zone (but less than 100%of the detection zone). In some instances, 100% of the detection zone isused for detection by a detector (e.g., detection in a transparentchannel by the unaided eye). The area in which the detection zone isused as an optical detection pathway may also depend on, for instance,the opacity of the material in which the channel is fabricated (e.g.,whether all, or, a portion, of the channel is transparent), the amountof a non-transparent material that may cover a portion of the channel(e.g., via use of a protective cover), and/or the size of the detectorand the detection zone.

In one embodiment, a signal produced by the reaction is homogenous overthe entire detection zone (e.g., over an entire meandering channelregion). That is, the detection zone (e.g., meandering channel region)may allow production and/or detection of a single, homogenous signal insaid region upon carrying out a chemical and/or biological reaction(e.g., and upon detection by a detector). Prior to carrying out areaction in the meandering channel region, the meandering channel mayinclude, for example, a single species (and concentration of species) tobe detected/determined. The species may be adsorbed to a surface of themeandering channel. In another embodiment, the signal may be homogeneousover only portions of the meandering region, and one or more detectorsmay detect different signals within each of the portions. In certaininstances, more than one detection zone can be connected in series andeach detection zone can be used to detect/determine a different species.

In some embodiments, a chemical and/or biological reaction involvesbinding. Different types of binding may take place in devices describedherein. The term “binding” refers to the interaction between acorresponding pair of molecules that exhibit mutual affinity or bindingcapacity, typically specific or non-specific binding or interaction,including biochemical, physiological, and/or pharmaceuticalinteractions. Biological binding defines a type of interaction thatoccurs between pairs of molecules including proteins, nucleic acids,glycoproteins, carbohydrates, hormones and the like. Specific examplesinclude antibody/antigen, antibody/hapten, enzyme/substrate,enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrierprotein/substrate, lectin/carbohydrate, receptor/hormone,receptor/effector, complementary strands of nucleic acid,protein/nucleic acid repressor/inducer, ligand/cell surface receptor,virus/ligand, etc.

In some cases, a heterogeneous reaction (or assay) may take place in achannel; for example, a binding partner may be associated with a surfaceof a channel, and the complementary binding partner may be present inthe fluid phase. The term “binding partner” refers to a molecule thatcan undergo binding with a particular molecule. Biological bindingpartners are examples; for instance, Protein A is a binding partner ofthe biological molecule IgG, and vice versa. Likewise, an antibody is abinding partner to its antigen, and vice versa. In other cases, ahomogeneous reaction may occur in the channel. For instance, bothbinding partners can be present in the fluid phase (e.g., in two-fluidlaminar flow system). Non-limiting examples of typical reactions thatcan be performed in a meandering channel system include chemicalreactions, enzymatic reactions, immuno-based reactions (e.g.,antigen-antibody), and cell-based reactions.

In another embodiment of the invention, a microfluidic device developedto perform a specific clinical test is labeled with information specificto the test (e.g., name of the test, batch-specific data and expirationdate). One or more components of the system, such as the sampleintroduction component, can be designed such that it is marked withpatient-specific information (e.g., physically or electronically). Uponattachment of the sample introduction component to a microfluidic device(e.g., a microfluidic substrate, optionally in connection with other(e.g., electronic) components), the patient's information can becomelinked to the device and the particular test performed on the device. Insome cases, e.g., for certain embodiments involving permanent attachmentof the sample introduction component to a disposable microfluidic device(e.g., by zip tie or snapping mechanism as described above), the twosets of information (one from the sample introduction component and onefrom the microfluidic device) cannot be separated. This can provide asafe method for adding the patient's information onto the microfluidicdevice. For example, in one embodiment, a microfluidic device is labeledwith test-specific information (e.g., name of the test, data for thetest calibration, batch name and number), and the sample introductioncomponent includes a surface that can accommodate a standard-sizedsticker containing a code referring to the patient identity (e.g., a barcode).

Though in some embodiments, systems of the invention may bemicrofluidic, in certain embodiments, the invention is not limited tomicrofluidic systems and may relate to other types of fluidic systems.“Microfluidic,” as used herein, refers to a device, apparatus or systemincluding at least one fluid channel having a cross-sectional dimensionof less than 1 mm, and a ratio of length to largest cross-sectionaldimension of at least 3:1. A “microfluidic channel,” as used herein, isa channel meeting these criteria.

The “cross-sectional dimension” (e.g., a diameter) of the channel ismeasured perpendicular to the direction of fluid flow. Most fluidchannels in components of the invention have maximum cross-sectionaldimensions less than 2 mm, and in some cases, less than 1 mm. In one setof embodiments, all fluid channels containing embodiments of theinvention are microfluidic or have a largest cross sectional dimensionof no more than 2 mm or 1 mm. In another set of embodiments, the maximumcross-sectional dimension of the channel(s) containing embodiments ofthe invention are less than 500 microns, less than 200 microns, lessthan 100 microns, less than 50 microns, or less than 25 microns. In somecases the dimensions of the channel may be chosen such that fluid isable to freely flow through the article or substrate. The dimensions ofthe channel may also be chosen, for example, to allow a certainvolumetric or linear flowrate of fluid in the channel. Of course, thenumber of channels and the shape of the channels can be varied by anymethod known to those of ordinary skill in the art. In some cases, morethan one channel or capillary may be used.

A “channel,” as used herein, means a feature on or in an article(substrate) that at least partially directs the flow of a fluid. Thechannel can have any cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least 2:1, more typically at least 3:1, 5:1, or 10:1 ormore. An open channel generally will include characteristics thatfacilitate control over fluid transport, e.g., structuralcharacteristics (an elongated indentation) and/or physical or chemicalcharacteristics (hydrophobicity vs. hydrophilicity) or othercharacteristics that can exert a force (e.g., a containing force) on afluid. The fluid within the channel may partially or completely fill thechannel. In some cases where an open channel is used, the fluid may beheld within the channel, for example, using surface tension (e.g., aconcave or convex meniscus).

A microfluidic substrate can be fabricated of any material suitable forforming a microchannel. Non-limiting examples of materials includepolymers (e.g., polyethylene, polystyrene, polycarbonate,poly(dimethylsiloxane), and a cyclo-olefin copolymer (COC)), glass,quartz, and silicon. Those of ordinary skill in the art can readilyselect a suitable material based upon e.g., its rigidity, its inertnessto (e.g., freedom from degradation by) a fluid to be passed through it,its robustness at a temperature at which a particular device is to beused, and/or its transparency/opacity to light (e.g., in the ultravioletand visible regions). In some embodiments, the material and dimensions(e.g., thickness) of a substrate are chosen such that the substrate issubstantially impermeable to water vapor.

In some instances, an microfluidic substrate is comprised of acombination of two or more materials, such as the ones listed above. Forinstance, the channels of the device may be formed in a first material(e.g., poly(dimethylsiloxane)), and a cover that is formed in a secondmaterial (e.g., polystyrene) may be used to seal the channels. Inanother embodiment, a channels of the device may be formed inpolystyrene or other polymers (e.g., by injection molding) and abiocompatible tape may be used to seal the channels. A variety ofmethods can be used to seal a microfluidic channel or portions of achannel, including but not limited to, the use of adhesives, gluing,bonding, or by mechanical methods (e.g., clamping).

The following examples are intended to illustrate certain embodiments ofthe present invention, but are not to be construed as limiting and donot exemplify the full scope of the invention.

Example 1 Fabrication of Microfluidic Channels in a Substrate

A method for fabricating a microfluidic channel system is described.

The layouts of the channel system were designed with a computer-aideddesign (CAD) program and are illustrated in FIGS. 3 and 4. Themicrofluidic devices were formed in poly(dimethylsiloxane) Sylgard 184(PDMS, Dow Corning, Distrelec, Switzerland) by rapid prototyping usingmasters made in SU8 photoresist (MicroChem, Newton, Mass.). The masterswere produced on a silicon wafer and were used to replicate the negativepattern in PDMS. The masters contained two levels of SU8, one level witha thickness (height) of ˜50 μm defining the channels in the immunoassayarea, and a second thickness (height) of ˜250 μm defining the reagentstorage and waste areas. The master was silanized with(tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane (ABC-R, Germany).PDMS was mixed according to the manufacturer's instructions and pouredonto the master. After polymerization (4 hours, 65°), the PDMS replicawas peeled off the master and access ports were punched out of the PDMSusing brass tubing with sharpened edges (1.5 mm in diameter). Tocomplete the fluidic network, a flat substrate such as a glass slide,silicon wafer, polystyrene surface, flat slab of PDMS, or an adhesivetape was used as a cover and placed against the PDMS surface. The coverwas held in place either by van der Waals forces, or fixed to the PDMSusing an adhesive.

In other embodiments, the microfluidic channels were made in polystyreneby injection molding. This method is known to those of ordinary skill inthe art.

Example 2 Storing Reagents in a Microfluidic System

This example describes a method for storing dry and liquid reagents in amicrofluidic system.

Dry and wet reagents were stored in the microfluidic systems shown inFIGS. 3-5 and 14. To store dry reagents, drops of biomolecules wereplaced onto the detection zones of the substrate. After 30 min, thesolution was removed and the surface of the substrate modified withproteins was rinsed with buffer. The surface was dried with compressednitrogen for 20 s, and then the substrate was sealed against a cover.The cover was either a plate of polystyrene (in the case PDMSsubstrates) (NUNC Omnitray, VWR, Switzerland) or a biocompatibleadhesive (in the case of polystyrene substrates). When a biocompatibleadhesive was used, the polystyrene substrate was drilled to obtainaccess holes prior to application of the cover. In a different approach,the holes were formed in the thermoplastic during the injection moldingprocess by using pillars inside the cavity of the injection moldingmachine. All of the microchannels, including those of the reagentstorage and immunoassay areas, were filled with blocking buffer (Tween20 and/or BSA in phosphate buffered saline (PBS)) to render the surfacesof the microfluidic channels hydrophilic and to block the surfaces toavoid non-specific adsorption of protein on the walls of themicrochannels. The blocking solution was removed by suction and thedevice was dried at room temperature under vacuum.

To store wet reagents in the microfluidic system, reagent solutions foran immunoassay were first prepared in separate containers (e.g., wellsof a 96-well plate, or centrifuge tubes). The reagents were sequentiallyaspirated as liquid plugs, followed by air spacers between successiveliquid plugs, into secondary tubing (polyethylene with a inner diameterof 0.2 mm) with a manually operated syringe connected to the back of thetubing.

Reagents were stored in channels of a reagent storage area of themicrofluidic system (fabricated by the method described in Example 1) byconnecting an outlet port of the tubing into an inlet of the channel.The fluids flowed from the tubing to the channel by either capillaryforces, applying negative pressure (e.g., a vacuum) to the outlet of thechannel, or by applying positive pressure to the inlet of the tubing(using a syringe plunger). The reagents resided in the reagent storagearea of the channel.

The inlets and outlets of the channels were then sealed by placing abiocompatible adhesive over the inlets and outlets. In the case of apolystyrene substrate, this second tape was applied onto the surfaceopposite of the surface modified with the cover. This sealing protectedthe stored reagents from degradation/denaturation due to atmosphericconditions.

The reagents were stored in the microfluidic channels for three monthswithout degradation/denaturation, as tested by use of the reagents inquantitative immunoassays. This example shows that both dry and liquidreagents (including proteins) can be stored for extended periods of timein microfluidic channels.

Example 3 Performing an Immunoassay by Loading a Sample Using anOpen-Ended Capillary Tube

This example shows that an immunoassay can be performed by loading asample using an open-ended capillary tube and using reagents stored on amicrofluidic substrate.

The microfluidic system of FIG. 7 was fabricated using the methoddescribed in Example 1. This system included four sections: a reagentstorage area, a sample loading area, an immunoassay area and a wastearea. The reagent storage area was pre-filled with reagents required toperform an immunoassay for the detection of total human IgG in wholeblood: antibody solutions, washing buffers and amplification reagents(either enzymatic substrates or silver amplification reagents) using themethod described in Example 2. These reagents were presented asready-to-use aqueous solutions loaded as a sequence of liquid plugs,separated from each other by air gaps.

A sample of blood from a donor was obtained and the sample was loadedinto a capillary tube (e.g., as shown in FIG. 8A) by capillary forces(or, in other experiments, by aspirating the sample in the capillarytube using a negative pressure applied at the other end of the tube).The outlet of the capillary tube was fitted to the sample loading portof the substrate and the sample was introduced into the microfluidicsystem by moving the frit within the tube towards the end of thecapillary tube with a plunger. Because the reagent inlet of themicrofluidic substrate had been previously sealed, the flow of samplewas automatically directed inside the microfluidic channel towards theoutlet of the device, which was vented. The capillary was left in place,and the frit (now wetted with sample) acted as an air-tight seal.

After introducing the sample into the substrate, the seal over the inletand outlet ports were removed. Application of vacuum at the outlet ofthe system resulted in the delivery of the sample and the reagents tothe immunoassay area according to the sequence pre-defined by the orderof reagents lined up inside the reagent storage area. All fluids exitingthe immunoassay area were eventually trapped inside the waste area.After completion of the assay, a signal specific for the target analytewas observable in the immunoassay areas.

Example 4 Performing an Immunoassay by Loading a Sample Using a FluidicConnector

This example shows that an immunoassay can be performed by loading asample using a fluidic connector and using reagents stored on amicrofluidic substrate.

The microfluidic system of FIG. 5 was fabricated using the methoddescribed in Example 1. Device 300 includes sections 302 containing wetstored reagents and section 350 containing stored dry reagents.Immunoassay area 360 was pre-fabricated with physisorbed molecules usingthe method described in Example 2. The immunoassay area included a firstdetection zone 362 patterned with Tween (using a solution of Tween inPBS), second and third detection zones 364 and 366 patterned withanti-human IgG (using a solution of anti-human IgG in PBS), and a fourthdetection zone 368 included patterned human IgG (using a solution ofhuman IgG in PBS).

Reagent storage area 304 was pre-filled (using the method described inExample 2) with reagents required to perform an immunoassay for thedetection of total human IgG in whole blood. The reagents were filled inthe form of a sequence of liquid plugs, each of the liquid plugsseparated by gaseous spacers. The reagents in lower portion 306 of thereagent storage area were (in order of introduction into the immunoassayarea): three buffer washes, one plug of anti-human IgG labeled with goldcolloid, three buffer washes, and six water washes. Upper portion 305 ofthe reagent storage area contained solutions for electroless silverdeposition used as the amplification solutions. These solutions includedsilver salt, stored in channel 308, and hydroquinone, stored in channel309. These solutions were kept separate prior to use. In FIG. 5A, theinlet 354 and outlet 318, which had previously been sealed, wereunsealed at this stage.

A sample of venous blood from a healthy donor was obtained and thesample was loaded into a fluidic connector by capillary forces (or, inother experiments, by aspirating the sample in the capillary tube usinga negative pressure applied at the other end of the tube). The fluidicconnector was filled with a known, predetermined volume of sample (15μL) by choosing an appropriate length of the capillary (and knowing theinternal volume of the capillary). (This volume of sample was enough tosustain sample incubation for 10 minutes after the source of vacuum wasset at −15 kPa.) The fluidic connector was bent so that one end of thefluidic connector fit into an outlet 318 of the reagent storage area,and the other end fit into an inlet 354 leading to the immunoassay area(see FIG. 5B). The fluidic connector enabled fluidic connection betweensections 302 and 350. In FIG. 5A, inlets 316 and 317 and outlet 356,which had previously been sealed, were unsealed at this stage.

Application of a source of vacuum 390 (−15 kPa) at outlet 356 of thesystem initiated the assay. The sample entered the immunoassay area,including detection zones 362, 364, 366, and 368 (FIG. 5C), followed bythe stored reagents from section 302 (FIG. 5D). The stored regents fromsection 302 included several rinsing reagents (e.g., buffer), whichwashed away any residual, unbound sample in the reaction area (FIG. 5D),as well as antibody solutions and amplification reagents.

After completion of the assay, an optical signal (a grayish film ofmetallic silver) specific for the analyte of interest was observable indetection zones 364, 366, and 368 of the immunoassay area (FIG. 5E).Using the series of physisorbed biomolecules in the detection zones asdescribed above, the following results were observed at the end of theassay: 1) no signal in the detection zone modified with Tween (adetergent known to prevent adhesion of proteins), as this detection zoneacts as an internal negative reference (detection zone 362); 2) aconcentration-dependant signal in the detection zones modified withanti-human IgG, reflecting the binding of human IgG from the sample(detection zones 364 and 366); and 3) a constant signal in the detectionzone modified with human IgG, which acts as an internal positivereference (detection zone 368). These observations were expected.

As shown in FIG. 5F, after removal of the fluidic connector and thesource of vacuum, the signal remained permanently bound in theimmunoassay area of the device, and could be directly observed and usedfor data storage.

This example demonstrates that a microfluidic system having storedreagents contained therein, connected by a fluidic connector containinga sample, can be used to detect total human IgG in a sample of wholeblood.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method comprising: providing a microfluidicsystem comprising a first microfluidic channel including at least oneinlet and one outlet and a second microfluidic channel including atleast one inlet and one outlet, wherein the first microfluidic channelcontains a first reagent disposed therein, wherein the secondmicrofluidic channel contains a second reagent disposed therein, andwherein a seal covers the inlet of the first microfluidic channel and aseal covers the outlet of the first microfluidic channel so as to storethe first reagent in the first microfluidic channel; and connecting afluidic connector comprising a fluid path to the microfluidic system tocause fluid communication between the first and second microfluidicchannels, wherein upon connection, the fluid path of the fluidicconnector connects to an inlet or an outlet of the first microfluidicchannel to allow fluid communication between the fluid path and thefirst microfluidic channel, and the fluid path connects to an inlet oran outlet of the second microfluidic channel to allow fluidcommunication between the fluid path and the second microfluidicchannel, wherein the first and second microfluidic channels are not influid communication with one another absent connection via the fluidicconnector.
 2. A method as in claim 1, wherein the microfluidic systemcomprises a reaction area in fluid communication with the secondmicrofluidic channel prior to the connecting step.
 3. A method as inclaim 1, wherein the fluid path includes a fluid path inlet and a fluidpath outlet, and wherein upon connection, the fluid path inlet connectsto the outlet of the first microfluidic channel to allow fluidcommunication between the fluid path and the first microfluidic channel,and the fluid path outlet connects to the inlet of the secondmicrofluidic channel to allow fluid communication between the fluid pathand the second microfluidic channel.
 4. A method as in claim 1, whereinthe first reagent disposed in the first microfluidic channel is a liquidreagent.
 5. A method as in claim 1, wherein the second reagent disposedin the second microfluidic channel is a substantially dry reagent.
 6. Amethod as in claim 1, wherein the first microfluidic channel comprises athird reagent disposed therein.
 7. A method as in claim 6, wherein thethird reagent is a liquid reagent.
 8. A method as in claim 7, whereinthe first and third reagents are separated by a fluid that is immisciblewith said first and third reagents.
 9. A method as in claim 8, whereinthe fluid immiscible with the first and third reagents is a gas.
 10. Amethod as in claim 9, wherein at least one of the first and thirdreagents is a rinse solution.
 11. A method as in claim 1, wherein themicrofluidic system comprises a seal covering the inlet of the secondmicrofluidic channel and a seal covering the outlet of the secondmicrofluidic channel.
 12. A method as in claim 2, wherein the secondreagent is disposed in the reaction area of the second microfluidicchannel, and wherein the second reagent is a substantially dry reagent.13. A method as in claim 12, wherein the second reagent comprises atleast one of an antibody or an antigen.
 14. A method as in claim 12,wherein the second reagent is adsorbed to a surface of the secondmicrofluidic channel.
 15. A method as in claim 2, wherein the reactionarea comprises at least one meandering channel region.
 16. A method asin claim 15, wherein the reaction area comprises at least two meanderingchannel regions connected in series.
 17. A method as in claim 16,wherein each of the at least two meandering channel regions comprises achemical and/or biological species disposed therein that can undergo achemical and/or biological reaction.
 18. A method as in claim 17,wherein the chemical and/or biological reaction is a binding eventbetween at least two binding partners, and wherein at least one of thebinding partners comprises an antibody.
 19. A method as in claim 2,wherein the reaction area comprises at least one cross-sectionaldimension of less than 100 microns.
 20. A method as in claim 1, whereinthe microfluidic system comprises a substrate comprising more than onesubstrate layers that are mated to one another.
 21. A method as in claim1, wherein the fluidic connector comprises a substantially planarsubstrate comprising a channel formed in at least a portion of thesubstantially planar substrate.
 22. A method as in claim 1, wherein thefluidic connector comprises a monolithic device comprising a channelformed in at least a portion of the monolithic device.
 23. A method asin claim 1, wherein the fluidic connector comprises a tube.
 24. A methodas in claim 1, wherein the fluidic connector comprises a fluid pathhaving a volume between 1 μL and 1000 μL.
 25. A method as in claim 1,wherein the fluid path of the fluidic connector comprises a reagentdisposed therein.
 26. A method as in claim 25, wherein the reagentdisposed in the fluid path of the fluidic connector is a substantiallydry reagent that coats at least a portion of the fluid path.
 27. Amethod as in claim 25, wherein the reagent is an anti-coagulant.
 28. Amethod as in claim 1, wherein the fluidic connector comprises a volumecontrol element that is constructed and arranged to introduce acontrolled volume of a fluid sample from a biological entity into thefluid path.
 29. A method as in claim 28, wherein the volume controlelement is constructed and arranged to allow a fluid sample to fill theentire volume of the fluid path.
 30. A method as in claim 28, whereinthe volume control element is constructed and arranged to allow a fluidsample to fill a portion, but not all, of the volume of the fluid path.31. A method as in claim 1, wherein the microfluidic system comprises asubstrate comprising the first and second microfluidic channels formedtherein.
 32. A method as in claim 1, comprising introducing a sampleinto the fluidic connector prior to the connection step.
 33. A method asin claim 32, wherein the fluidic connector contains a binding componentdisposed therein, the method comprising binding at least a portion ofthe sample with the binding component prior to the connecting step. 34.A method as in claim 32, comprising applying a vacuum to an outlet ofthe microfluidic system and flowing at least the sample and the firstreagent in series across an analysis region of the second microfluidicchannel.